Transcriptome analysis reveals fundamental differences in plant response to acute and chronic exposure to ionizing radiation

Available online at www.sciencedirect.com

Mutation Research 624 (2007) 101–113

Transcriptome analysis reveals fundamental differences 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 b

Department of Biological Sciences, University of Lethbridge, Lethbridge, Alta. T1K 3M4, Canada Institut de Biologie Mol´eculaire des Plantes, French National Center for Scientific Research (CNRS), 12, rue du g´en´eral Zimmer, 67000 Strasbourg, France c International Radioecology Laboratory, ICC, Post Box 151, Slavutych, Kiev, Ukraine Received 23 November 2006; received in revised form 7 April 2007; accepted 18 April 2007 Available online 5 May 2007

Abstract We analyzed the influence of acute and chronic ionizing radiation (IR) on plant genome stability and global genome expression. Plants from the “chronic” group were grown for 21 days on 137 Cs-artificially contaminated soil, and received a cumulative dose of 1 Gy. The “acute” plant group was exposed to an equal dose of radiation delivered as a single pulse. Analysis of homologous recombination (HR) events revealed a significantly higher increase in HR frequency (HRF) in the “chronic” group as compared to “acute” group. To understand the observed difference we performed global genome expression analysis. RNA profiling at 2 h and 24 h after acute irradiation showed two-third of up- and down-regulated genes to be similarly regulated at both time points. In contrast, less than 10% of the genes up- or down-regulated at 2 h or 24 h post-acute irradiation were similarly changed after chronic exposure. Promoter analysis revealed substantial differences in the specific regulatory elements found in acute and chronic transcriptomes. Further comparison of the data with existing profiles for several stresses, including UVC and heavy metals, showed substantial transcriptome similarities with the acute but not the chronic transcriptome. Plants exposed to chronic but not acute radiation showed early flowering; transcriptome analysis also revealed induction of flowering genes in “chronic” group. © 2007 Elsevier B.V. All rights reserved. Keywords: Ionizing radiation; Acute and chronic exposure; Arabidopsis; Genome stability; Global genome expression

1. Introduction The ionizing radiation (IR) causes a variety of DNA damages, including base and sugar alterations, formation ∗ Corresponding author. Tel.: +1 403 394 3916; fax: +1 403 329 2242. E-mail address: [email protected] (O. Kovalchuk).

0027-5107/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2007.04.009

of DNA–DNA and DNA–protein cross-links, as well as single-strand breaks (SSBs) and double-strand breaks (DSBs). It is, however, generally accepted that the DSBs are the main, if not the only type of damage that leads to the cell death [1]. Generated strand breaks are repaired by two major mechanisms, non-homologous end-joining (NHEJ) and homologous recombination (HR). IR is known to induce HR frequency (HRF), although it is not

102

I. Kovalchuk et al. / Mutation Research 624 (2007) 101–113

clear whether it is due to the increase of strand breaks or due to the stimulation of the activity of the repair enzymes. The response to IR varies dramatically among plants. The general effect on growth and development ranges from stimulatory effects at very low doses to increasingly harmful effects for vegetative and reproductive tissue at high radiation levels. The degree of the radiation effects is dependent on the species, age, plant morphology and physiology, and genome size and composition [2]. Woody plant species, in general, tend to be less resistant to IR as compared to herbaceous species [2]. The best known example is the pine Pinus silvestris, where an acute exposure to 60 Gy of IR resulted in the death of pine stands near the Chernobyl Nuclear Power Plant [3]. However, the lethal dose for Arabidopsis thaliana exceeds 100 Gy [4]. Similar doses of IR obtained from different types of radiation also trigger substantially different effects on the same plants. Several types of radiation, including ␤-, ␥-, and fast neutrons have been tested on soybean seedlings. ␤- and ␥-irradiation caused similar effects, while fission neutrons were 6–15 times more damaging [2]. In comparison to X-rays, irradiation of A. thaliana seeds with an equal dose of fast neutrons was shown to be nearly 10 times more efficient in causing semi-sterility [5]. A number of different plant-based assays exist to study DNA damage by radiation. Acute IR led to increased incidences of chromosome aberrations in Allium cepa [6], decreases in the initial cell number in A. thaliana embryos [7], increases in the frequency of chlorophyll deficient embryonic mutations [8] and of the frequency of homologous recombination events in Arabidopsis plants [9,10], as well as in the increase in appearance of recessive phenotypes in Tradescantia species [11]. Similarly, the exposure to chronic radiation was also shown to have a significant influence on plant morphology and genome integrity. For example, 60 Co ␥-rays increased the frequency of “pink” mutations in Tradescantia [12]. Further, ionizing radiation from Chernobyl exclusion zone increased the level of embryo lethal mutations in Arabidopsis [13], increased the recombination frequency and number of strand breaks in Arabidopsis plants [14], caused a number of morphogenetic changes in 96 different plant species [15], as well as induced an extremely high frequency of chromosomal aberrations in rye and wheat plants [16]. There is substantial amount of information accumulated on the influence of stress on plant transcriptome profile [17–22]. We recently reported the whole genome expression of plants exposed to Cd and Pb as well as to such mutagens as bleomycin, UVC, and xylanase

[17,18]. However, a comparison of acute and chronic ionizing radiation stress on plant transcriptome is missing. We have previously found that the chronic exposure to the low dose of IR had a more pronounced effect on recombination than the acute exposure. To understand the changes occurring in plants exposed to chronic or acute IR, we repeated the analysis of HRF and profiled the global genome expression. Our data confirmed the stronger HRF increase in chronically exposed plants and showed drastic differences in their transcriptome. The comparison of the “acute” and “chronic” IR-induced transcriptomes with transcriptomes of the other stresses revealed substantial similarities between “acute” IR transcriptome and transcriptomes of other stresses. In contrast, the “chronic” transcriptome was rather unique and had no similarities to any other transcriptome. 2. Methods 2.1. Plant growth, radiation exposure and sampling Plants of transgenic line #11 that carried in the genome single copy of truncated ␤-glucuronidase gene [23] were planted on clean soil or on soil artificially polluted with 137 Cs. Plants were grown at 22 ◦ C at a 16 h/8 h day/night regime. Plants from acute group were irradiated with 60 Co at 21st day (for the microchip experiment) or at 14th day (for recombination measurement) after germination. Irradiation was done using 60 Co gun (Agat-R1), 0.025 Gy/s to the total dose of 1 Gy. Plants from chronic group were grown on soddy-podsolic soil artificially polluted with a standard solution of 137 CsCl to the final sample activity of 34 MBq/kg (±10%). The sample activity was calculated based on our previous experiments with this soil. Previous data revealed that Arabidopsis plants grown for 35 days on the soil with the activity of 4 kBq/kg absorbs the total dose of 199.2 ␮Gy, with the contribution of internal dose being −24% [10]. To obtain the total absorbed dose of 1 Gy within shorter period of time (21 days), one would need 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 was mock treated. Approximately 300 plants were planted for each treatment, and the experiment was performed three times. The actual absorbed dose for plants was calculated as the sum of the external and internal dose [24]. The gammairradiation exposure rate (R/h) in the air at the soil level was determined using a SRB9-1 ship beta–gamma radiometer, a FD-5 field dosimeter and a DC9-04 dosimeter control signal. External dose as total ␥-dose per day was calculated from readings. The internal dose is a consequence of uptake of 137 Cs by plants. 137 Cs content in plant tissues was determined by spectrometric and radiochemical methods. The radiation trans-

I. Kovalchuk et al. / Mutation Research 624 (2007) 101–113

fer factor for A. thaliana plants was calculated as relationship between concentration of 137 Cs (Bq/kg of dry weight) in plants to the concentration of 137 Cs in soil (kBq/m2 ), taking soil properties into consideration, as described [25]. The two different radionuclide used for the experiment, 60 Co and 137 Cs, have comparable radiological characteristics. Both are ␤- and ␥emitters and have similar equivalent doses. The dose rate at a depth of 0.07 mm in tissue (directional equivalent dose rate) at a distance of 10 cm from a source of radiation with an activity of 1 GBq (109 ) is 1000 mSv/h for 60 Co and 2000 mSv/h for 137 Cs. A surface contamination of 1 kBq/cm2 leads to the dose of 1.1 mSv/h for 60 Co and 1.5 mSv/h for 137 Cs. 2.2. Histochemical staining procedure Histochemical staining was done according to Jefferson [26]. For destructive staining plants were vacuum infiltrated 2× 10 min in sterile staining buffer containing 100 mg 5-bromo4-chloro-3-indolyl glucuronide (X-glu) substrate (Jersey Labs Inc., USA). Afterwards plants were incubated at 37 ◦ C during 48 h and bleached with ethanol. 2.3. RNA preparation and microchip hybridization Total RNA was isolated from control, acutely (2 h and 24 h) and chronically exposed Arabidopsis tissue using Trizol reagent (Life Technologies) following the supplier’s protocol. RNA was then further purified using the RNeasy total RNA clean up protocol (Qiagen). The integrity of the RNA samples was assessed by running an aliquot of samples on RNA 6000 Nano LabChip (Agilent) using the 2100 bioanalyzer (Agilent). Probe synthesis, hybridization to Affymetrix ATH1-121501 arrays and scanning were done according to Affymetrix protocol. Statistical analyses of the scans were done with the help of the Kensington Discovery Edition version 1.8 (Inforsense). Mean values of gene expression were calculated for each group of three RNA samples (three independent RNA hybridizations) prepared from 20 plants each. The expression values of the treatment groups were related to the respective controls and significance of the differences between the mean expression values was assessed using a Student’s t-test, two-tailed, paired. For further in depth analysis we selected those genes that had significantly (p < 0.05) increased or decreased their expression by more than 3-fold. 2.4. RNA preparation and reverse transcription The total RNA samples used for the real-time PCR were prepared exactly the same way, from control, acutely and chronically treated Arabidopsis tissue. The samples were treated with DNase I (Invitrogen) according to manufacturer’s instructions. After DNase inactivation the samples were purified with RNeasy Mini-columns (Qiagen). The RNA yields were measured using RiboGreen assay (Molecular Probes). Using 1.0 ␮g of the purified RNA as a template, reverse transcription was performed in a total volume of 33 ␮L for 1 h at

103

37 ◦ C according to manufacturer protocol (You-Prime-FirstStrand Kit, Amersham, UK). 2.5. Real-time PCR analysis The following genes and primers were used for realtime PCR: cor15a precursor (At2g42540) (+5 -atggcgatgtctttctcagg-3 ; −5 -acgacgaactgagttttctgg-3 ), MYB-related transcription factor CCA1, LHY (At2g46830), alternative NADH-dehydrogenase (256057 at), DNA-binding protein similar to CCA1 (At1g01060) (+5 -gaagaattattagctaaggc-3 ; −5 -atgttcttcaattcgttgcc-3 ), putative protein (At3g54500) (+5 -tttggaacaagatgattctgg-3 ; −5 -ctgcactaccagccaaccgg-3 ), DNAJ protein (At5g23240) (+5 -tctccgacgactcttcctcc-3 ; −5 -atcaaagtcagtgatagacg-3 ), DREB2A (At5g05410) (+5 -atggcagtttatgatcagag-3 ; −5 -ctcgttatactctttccatc-3 ), transcriptional activator CBF1, similar DREB1A (At1g12610) (+5 -aacgccgcatttggctcggg-3 ; −5 -tccggatcattggattccgg-3 ), At14a-1 (At3g28290) (+5 -atatggagaagtagtgtggg-3 ; −5 -tttttcaaactgtgccacgg-3 ), putative protein ethylene-responsive element binding protein homolog (At4g34410) (+5 -aacagaaccgaattcgtcgg-3 ; −5 -ctggaaaaaccctgacacgg-3 ). Real-time PCR was performed according to previously published protocols [17]. The actin RNA was used as a control, the actin primers sequence was: sense 5 -ACTGGCATGGCCTTCCG-3 , antisense 5 -CAGGCGGCACGTCAGATC-3 .

3. Results and discussion 3.1. Experimental set-up First, we analyzed the homologous recombination frequency 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 in 25 cm × 50 cm × 5 cm trays (Fig. 1A). The “chronic” group was germinated and grown in the same type of soil, but artificially polluted with 137 Cs. The total dose absorbed by chronically exposed plants was 0.93 ± 0.22 Gy and consisted of internal dose of 0.71 ± 0.21 Gy and external dose of 0.22 ± 0.03 Gy. The plants of the “acute” group received 1.0 Gy at 14 days post-germination, whereas “control” group were mock-irradiated. HRF was measured at 21 days post-germination. Second, we performed a global genome expression analysis of the “control”, “acute” and “chronic” tissue. For this experiment, three similar groups, “control”, “acute” and “chronic” were germinated and grown in pots with clean or contaminated soil. Each group consisted of 50 plants (Fig. 1B). Plants from “chronic” group were grown in polluted soil for 21 days, whereas plants from “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 recombination analysis. “Control” group was mock treated; “chronic” group was grown on contaminated soil and received 0.93 Gy; “acute” group was irradiated at day 14 after germination, obtaining 1 Gy. All plants were harvested for recombination analysis at day 21. Recombination substrate consists of two non-functional truncated overlapping copies of 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” group was irradiated at day 21 post-germination. Twenty plants from each experimental group were pooled together for RNA preparation and microchip analysis. Plants from acutely irradiated group were collected twice, at 2 h at 24 h after irradiation.

soil for 21 days and then were ␥-irradiated or mockirradiated, respectively. RNA samples were collected from all groups, pooling 20 plants together. “Acute” samples were collected at two different time points, 2 h and 24 h post-irradiation (Fig. 1B). 3.2. Chronic exposure to IR resulted in a substantially higher HRF increase than acute exposure HRF was found to be increased in both groups of plants, acutely and chronically treated. On average, the control plants had 1.25 ± 0.13 recombination events per plant, whereas acutely and chronically treated 2.57 ± 0.19 and 4.33 ± 0.36, respectively (Fig. 2). Further, the differences between HRFs in all groups were all statistically significant (single factor ANOVA, α = 0.01, p < 0.01 in all cases). Several papers suggest that the majority of DNA damage events are the result of the non-targeted influence of radiation on DNA [9,10,27–29]. The non-targeted events represent the variety of activities in the cell that were triggered by the response of irradiated cells. These activities could include the generation of free radicals, the production of small molecules, metabolites, short peptides or small regulatory RNAs capable of traveling

Fig. 2. Plants exposed to chronic radiation exhibited more pronounced increase in the frequency of homologous recombination. Y-axis shows the number of spots per plant, and X-axis shows the treatment group. All data points (diamonds), the mean (small black box inside of the white box), the maximum and minimum values (− sign), the 5–95% confidence interval (bars), 1–99% confidence interval (×) as well as S.D. (boxes) were calculated from three independent experiments.

from treated tissue and leading to the generation of nontargeted events such as DNA damage or activation of certain DNA repair mechanisms. On the other hand, it is simply possible that chronic radiation results in constant production of radicals that are under threshold of the activation of scavenging enzymes. In this case it would lead to more frequent and constant DNA damage, leading to regular formation of DSBs. We measured the level of DSBs and found most of the breaks to be repaired already at 6 h after acute radiation. In contrast, plants grown on contaminated soil exhibited higher level of DSBs as compared to control plants at any given time during the plant growth (data not shown). This implies that radiation triggers a series of events resulting in increased genome instability. Since a substantial and statistical difference in the level of HR events in “acute” and “chronic” groups was found, it was possible that there was also a substantial difference on the level of global genome expression. 3.3. Transcriptome analysis reveals significant overlap between 2 h and 24 h acute groups and almost no overlap with chronic group The RNA expression profiling consisted of three “control”, three “chronic”, six “acute” (2 h and 24 h time points) samples. The average data for plant tissue exposed to acute (2 h and 24 h) or chronic radiation were related to the average data for control plants. After the two cut-offs, a 3-fold change in activity (up- or downregulation) 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 the number of the genes that are uniquely (inside of the non-overlapping part) or commonly (inside of overlapping part) regulated. Left diagram shows the genes that change their activity by more than 3-fold, where “2h 3fold I”, “24 3fold I” and “ch 3fold I” were induced and “2h 3fold D”, “24 3fold D” and “ch 3fold D” were down-regulated genes. Total number of genes showing “response” were 20,683 and 21,225 for induced and down-regulated, respectively.

number of up- and down-regulated genes for each group was obtained. Our analysis revealed 449 up-regulated and 423 down-regulated genes in plants harvested 2 h after acute exposure and 237 up-regulated and 256 downregulated genes in plants harvested 24 h after acute exposure. Chronic radiation resulted in up-regulation of 138 and down-regulation of 139 genes (Fig. 3). Cross comparison between these three groups showed substantial similarity between “2 h” and ‘24 h” groups of plants and very little between “chronic” group and either of acute. It was found that 60–70% of all genes that were found to be up- or down-regulated 2 h after acute irradiation remained up- or down-regulated at 24 h (166 out of 237 and 161 out of 254 up- or down-regulated genes, respectively). However, plants from the “chronic” group had only about 10% of genes regulated commonly with either of “acute” groups (19 and 7 out of 138 up-regulated genes and 9 out of 139 down-regulated genes; Fig. 3). These data suggest that most of the genes that change their expression 2 h after acute exposure stay up- or down-regulated even 24 h after exposure; however, the degree of the regulation may change (see below). The fact that there was only a minor overlap between the “chronic” and “acute” groups suggest that different mechanisms are involved in the response to chronic or acute exposure to radiation. To test this hypothesis, upand down-regulated genes were grouped into several functional categories/protein groups. 3.4. Grouping the genes that changed their expression by the pathways revealed substantial differences To identify the genes/pathways influenced by acute and chronic exposure to radiation, we have grouped

up- and down-regulated genes into the following categories: “unknowns”, “cell wall associated”, “pathogen resistance”, “signal transduction”, “oxidative stress”, “hormone response”, “nucleic acid metabolism”, “transcription factors”, “general stress”, “sugar and lipid metabolism”, “calmodulins”, “protein and amino acid metabolism”, “transport”, “development and morphogenesis” and “not determined” (Fig. 4). The chronic group had the largest percentage of genes associated with “nucleic acid metabolism”, 6% and 11% for up- and down-regulated genes, respectively. In contrast the acute group had 3% up-regulated and 4% down-regulated genes in the 2 h group, and 1% up-regulated and 2% down-regulated genes in the 24 h group. The “nucleic acid metabolism” group included genes dealing with DNA repair, DNA binding (excluding the transcription factors), and DNA or RNA metabolism. The correlation between the higher frequency of HR and the substantially higher percentage of genes involved in nucleic acid metabolism in the chronic group could potentially suggest more frequent modification of their nucleic acids (DNA/RNA). Indeed, several putative transposons, retrotransposon, and reverse transcriptases were found to be down-regulated in the “chronic” group, while not one such gene changed expression upon acute exposure. The most pronounced difference was observed in the regulation of genes involved in signal transduction, where 10% and 7% of up- and down-regulated genes in the “acute 2 h” group and 20% and 12% in the “acute 24 h” group, compared to 5% and 3% in the chronic group were observed, respectively. This is not surprising as the acute exposure is a severe stress that changes the regulation of multiple pathways, and thus elaborates multiple signal transduction cascades.

106

I. Kovalchuk et al. / Mutation Research 624 (2007) 101–113

Fig. 4. Up- and down-regulated genes were grouped to various pathways. Up-regulated (A2 I, A24 I and Ch I) and down-regulated (A2 D, A24 D and Ch D) genes were distributed to 15 different groups, including genes with unknown function as well as genes involved in signal transduction, oxidative stress response, transport, etc.

The most well represented group of genes was the group 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 differences in regulation of several specific genes. For example, 20 different cytochrome P450 genes were up- and down-regulated in 2 h group, and 16 of them remained similarly changed at 24 h. In contrast, only three such genes changed their expression upon chronic exposure. Cytochrome P450 monooxygenases are involved in the biosynthesis of various compounds in plants such as phenylpropanoids, lipids, and phytohormones [30]. The CYP86A and CYP94B cytochrome P450 monooxygenase subfamilies are fatty acid omega-hydroxylases involved in the synthesis of cutin, production of signaling molecules, and prevention of accumulation of toxic levels of free fatty acids in plant cells liberated by phospholipases in early response to stress [31]. The fact that

we found changes in the expression of the monooxygenases primarily at 2 h and 24 h after the exposure supports the idea that they are mostly important in an immediate stress response. Kitahata et al. also showed the involvement of monooxygenases in the chemical regulation of abscisic acid catabolism [32], furthering this notion. Another group of oxidative stress-related genes that changed their expression were the peroxidases. We found pr10, ATP8a, ATP3a, ATP12a, ATP13a, ATP14a, ATP17a, ATP20a, ATP21a, and ATP23a to be downregulated at 2 h or 24 h after the acute exposure. However, in the chronic group, a single putative ATP2a-like peroxidase was down-regulated. It has previously been shown that the exposure of Arabidopsis to salt stress resulted in the down-regulation of several key peroxidases [33]. Moreover, it was found that antisense suppression of cytosolic tobacco ascorbate peroxidase resulted in higher tolerance to salt and heat stress [34], while the overexpression of ascorbate peroxidase in

I. Kovalchuk et al. / Mutation Research 624 (2007) 101–113

tobacco chloroplasts enhanced tolerance to salt stress and water deficit [35]. Further still, Arabidopsis plants overexpressing thylakoidal ascorbate peroxidase showed increased resistance to paraquat-induced photooxidative stress and to nitric oxide-induced cell death [36]. These studies all demonstrate that the regulation of peroxidases and other oxidative stress-related enzymes are key components of the response variety of stresses. Another group of oxidative stress-related genes differentially regulated by acute and chronic stress are the glutathione-S-transferases (GSTs). Ten and 11 different GSTs were up- and down-regulated at both the 2 h and 24 h post-acute exposure, respectively. At the same time, chronic exposure resulted in the change of a single GST gene. GSTs are known to be induced by various ROSdependent stresses [37], drought [38], and influence of ethylene and auxin [39]. Again, it is possible that the response that is triggered in plants by chronic stress does not reach a ‘threshold level’ for the regulation of the aforementioned oxidative stress-related genes, and thus they are not present in the microchip profile. The comparison of the “hormone response” group also revealed substantial differences. The acute 2 h group contained 8 ethylene-responsive binding factors and 5 auxin-responsive factors out of 17 up-regulated genes and as well as 6 auxin-responsive and 1 ethyleneresponsive genes out of 14 down-regulated “hormone response” genes. The acute 24 h group contained 4 auxin-responsive and 9 ethylene-responsive genes out of 13 “hormone-related” genes. In contrast, seven out of eight “hormone response” genes up-regulated by chronic stress were auxin-related. Recent research

107

papers describe the close link between the exposure to abiotic stress and auxin/ethylene regulation. Transcription factors operating downstream of ethylene and auxin have been shown to be responsive to salt stress [40]. Further, Mishra et al. has shown that stress signaling occurs not only through MAP kinases, but also through auxin and ethylene pathways [41], and it was previously shown that mechanical wounding regulates a number of auxin- and ethylene-responsive genes [42]. As chronic exposure to radiation induces vegetation growth, this might be in part due the (in)direct stimulation of auxin-responsive genes. In our experiments, we found that the exposure to chronic, but not to acute radiation, induced the putative flowering-time gene CONSTANS (At3g02380) by 31-fold. Chronic stress also induced several genes involved in photosynthesis and carbohydrate metabolism, starch synthase (11-fold; At1g32900), chlorophyll a/b binding protein 151 precursor (10-fold; At3g27690), and a putative chlorophyll a/b binding protein (3.4-fold; At2g05070). In contrast, acute stress down-regulated this group of genes at both 2 h and 24 h: sucrose synthase (56.0- and 26.6-fold; At3g43190), 6-phosphogluconolactonase-like protein (4.2- and 3.5fold; At1g13700), and glucose fermentation (3.1- and 3.2-fold; 255924 at). This further supports the idea that chronic stress induces vegetative growth, and that this is likely regulated via auxin-responsive genes. In contrast, acute exposures to stress are perceived as more immediate and severe, and thus result in an abrupt inhibition of most of the growth-related pathways. This is, however, a transient reaction that is normally reversible after several days.

Fig. 5. Plants exposed to chronic radiation flowered earlier. For the pilot experiment, three groups of plants (10 plants per each group) were exposed to acute or chronic radiation or mock-treated. Flowering time (Y-axis, in days) for individual plants (10 per group) was recorded in each group (X-axis) and averages were calculated. Similar letters show statistically identical results, whereas different letters show statistically significantly different results.

108

I. Kovalchuk et al. / Mutation Research 624 (2007) 101–113

As a support of the microchip findings, we found that plants exposed to chronic stress flowered on average 3 days earlier than plants belonging to either “acute” or “control” groups (Fig. 5). Several early reports have suggested that the chronic exposure to radiation stimulate flowering [43,44]. However, one report showed no significant changes in the flowering time of Arabidopsis chronically exposed to gamma-radiation [45]. Despite this, other works have shown the regulation of flowering time upon stress exposure. Daly and Thompson [45] showed that exposure of plants to drought stress positively influences the flowering time [46], while, more recently, Martinez et al. suggested that various stresses, such as light, drought, high temperatures, pathogen infection and exposure to UVC can influence the transition to flowering [47]. Several other stress-induced genes were influenced by exposure to radiation. Cold regulated proteins cor15a and cor15b were induced by more than 20-fold by both chronic and acute stress. DREB2A were induced by 14-, 12.1-, and 3.4-fold by acute (2 h and 24 h) and chronic stress, respectively. DREB2A was shown to be induced by water stress and regulating the expression of many water stress-inducible genes [48]. Similarly, genes DREB2B and DREB1C, commonly induced by various stresses, were also induced by acute radiation exposure by 3.3- and 11.4-fold for DREB2B and 3.9- and 16.9-fold for DREB1C at 2 h and 24 h, respectively. Another substantial difference observed between acute and chronic groups was the regulation of chromatin. CCR4-associated factor 1-like (CAF1, At3g44260) was induced only by acute stress (14.2and 21.3-fold at 2 h and 24 h, respectively). CCR4 and CAF1 are two catalytic subunits with deadenylase activity believed to be involved in DNA damage response in yeast [49]. Mulder et al. suggested that the induction of CAF1 by ionizing radiation in yeast is part of the general stress response [50]. Further, Westmoreland et al. showed that the yeast cell cycle transition through G1 and S phases is CCR4-dependent upon the exposure to stress [51]. Also regulated was GCN5-related N-acetyltransferase (GNAT, At2g39030). It was induced by 5.5-fold at 2 h post-acute exposure. GCN5 was associated with UV-radiation response and response to high light in yeast models [52,53]. 3.5. Comparison of the data for IR, UVC and Cd/Pb To further explore the differences in transcriptomics of acute and chronic groups, we compared the set of these genes to those regulated by UVC exposure and the exposure to heavy metals [17,18]. When we compared acutely

regulated genes, we found many similarities. There were 78 up-regulated and 15 down-regulated genes upon UVC exposure and 20 up-regulated and 47 down-regulated genes upon exposure to heavy metals, similarly regulated by acute exposure to IR (2 h sample; Supplementary Table 1). This calculated to a nearly 20% (78 out of 449) of all genes that were induced upon 2 h exposure to radiation to be also induced by UVC. Concurrently, 10% (47 out of 423) of all genes that were down-regulated at 2 h after IR exposure were down-regulated by exposure to heavy metals. Surprisingly, there was not a single gene that was regulated in both chronic exposure to IR as well as exposure to heavy metals or UVC. This comparison further confirms the “specificity” of chronic exposure. The above multi-stress analysis confirmed that exposure to chronic radiation represents a different type of stress when compared to acute exposure to IR and even exposure to UVC and heavy metals. The changes in transcriptome of the chronically exposed plants may represent an adaptation to a milder and constant type of stress. The higher increase in HRF in chronically exposed stress also supports this assumption. In animals, it is proposed that exposure to radiation leads to the shift from non-homologous to homologous repair of strand breaks [54,55]. This mechanism is especially apparent in dividing cells because they spend less time in the G1 phase, when HR repair is down-regulated [54,55]. This shift in the utilization of the strand break repair pathways may represent a protection mechanism directed to longterm survival. It seems logical that a similar mechanism may be conserved in plants. 3.6. Data comparison to previous publications Another important comparison we performed was the comparison to a set of data published by Nagata et al. [56]. They performed the microchip analysis of A. thaliana plants exposed to 2000 Gy of Co60 harvested at 2 h and 24 h post-irradiation. Comparison of the set of genes induced at 2 h post-irradiation revealed that half of the genes (14 out of 28) reported by Nagata et al. were also changed in our experiment [56] (Table 3S). In contrast, only 3 genes in our experiment out of the total 19 genes reported by Nagata et al. were also induced at 24 h post-irradiation [56] (Supplementary Table 2). The number 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 similarities in the transcriptomes in both experiments. The main reason could be that Nagata et al. exposed plants to the dose that was 2000-fold higher than the dose used

I. Kovalchuk et al. / Mutation Research 624 (2007) 101–113

in our experiment (2000 versus 1 Gy) [56], theoretically causing exponentially higher amounts of damage and stress. Additionally, differences in growth conditions of the plants could also have a substantial influence on the transcriptome. To objectively compare this data, experimental plants would have to be grown under identical conditions before the different doses of radiation were dealt. 3.7. Real-time PCR confirmation of the gene expression In order to confirm the validity of the microchip approach, we performed real-time PCR (RT-PCR) analysis of 10 genes commonly or differentially up-regulated by the acute and chronic exposure to IR. The following genes were analyzed: cold-regulated cor15a precursor (At2g42540), MYB-related transcription factor (At2g46830), alternative NADH-dehydrogenase (256057 at), DNA-binding protein similar to CCA1 (At1g01060), putative protein (At3g54500), DNAJ protein (At5g23240), DREB2A (At5g05410), transcriptional activator CBF1 (At1g12610), At14a-1 (At3g28290), putative protein ethylene-responsive element binding protein homolog (At4g34410). The RT-PCR confirmed the expression pattern observed in microchip experiment, although the degree of regulation was somewhat different (Table 1). Next, we analyzed the expression pattern of several of these genes using the database provided by Affymetrix (www.affymetrix.com) and found the majority of these genes to change their expression upon exposure to various stresses, including salt, mannitol, drought, cold, UVB, mitomycin and bleomycin. For example, cor15a (At2g42540) did not change its expression at 1 h after cold exposure, but did strongly

109

(>10-fold) at 24 h. It was strongly induced by a stream of dry air (at 1 h and 3 h after exposure), by the exposure to mannitol (3–24 h after exposure), and by the exposure to salt (3–24 h after exposure). At the same time 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 h after exposure, down-regulated by exposure to highlight (within the first 1 h), strongly down-regulated by exposure to UVB, drought, mannitol, salt, mitomycin and bleomycin (3–12 h). DREB2A (At5g05410) was induced 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-responsive element, CCA1 related were also similarly induced by number of stresses. The above-mentioned comparison confirms how similar the transcriptomes of A. thaliana plants exposed to variety of stresses are. This further suggests that it should be possible to select the group of common “stress transcriptome” as well as to identify the unique IDs of each specific stress. This, however, was beyond the scope of current report. 3.8. Promoter analysis We analyzed the promoter areas of genes belonging to “transcription factors and DNA binding”, “general stress response”, “nucleic acid metabolism”, “oxidative stress” and “signal transduction” for the following elements: ABA-responsive elements (ABRE; PyACGTGGC), dehydration-responsive elements (DRE; TACCGACAT), DRE-related core motif (core; CCGAC), ubiquitous regulatory elements (Gbox; ACCGTG), MYB (TAACGGTT and other multiple motifs), MYC (multiple motifs), sex-determining region

Table 1 Real-time PCR data Gene

2h

24 h

Chronic

cor15a precursor (At2g42540) MYB-related transcription factor CCA1, LHY (At2g46830) Alternative NADH-dehydrogenase (At1g07180) DNA-binding protein similar to CCA1 (At1g01060) Putative protein (At3g54500) DNAJ protein (At5g23240) DREB2A (At5g05410) Transcriptional activator CBF1 similar DREB1A (At1g12610) At14a-1 (At3g28290) Putative protein ethylene-responsive element binding protein homolog (At4g34410)

72.8(469.4) N/C(N/C) N/C(N/C) −4.1(−3.8) −2.2(−3.5) 8.3(77.5) 9.8(14.2) 17.2(100.5) 12.1(100.2) 9.3(49.8)

26.1(142.2) −4.9(−12.6) −3.1(−3.8) −5.9(−14.3) −4.8(−7.7) 17.6(95.3) 4.8(12.1) 9.1(76.3) 27.8(442.1) 15.2(67.7)

3.9(3.1) 10.5(26.7) 9.3(15.0) 9.4(16.2) 6.2(18.4) −42.6(−149.9) −2.9(−3.4) N/C(N/C) N/C(N/C) N/C(N/C)

The real-time PCR data is presented as a ratio to control. Original data was calculated as an average from four reactions (two independent runs from each of two independent RNA preparations). The microchip data is in parentheses. “N/C” stands for not changed.

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. Promoter areas (2000 nucleotides) of the genes belonging to “general stress”, “transcription factors”, “signal transduction” and “oxidative stress” were analyzed for the following motives: ABRE, DRE, G-box, MYB, MYC, core, Oct, SRY, HNF-3, CdxA. Y-axis shows the percentage of genes containing one of the motives, whereas X-axis shows different motives found in 2 I, 24 I, Ch I, 2 D, 24 D and Ch D groups.

Y (SRY; AAACAAA), CdxA, chicken homeobox gene TF (CdxA; ATTAATA), octamer protein binding sites/X-ray-responsive elements (Oct; ATGCAAAT), and hepatocyte nuclear factor/X-ray-responsive elements (HNF-3; GTTTGTTTT). A detailed analysis of 2000 nts of the promoter region revealed substantial differences in distribution of the elements among the genes belonging to “acute” or “chronic” groups (Fig. 6). At the same time, no difference was found upon comparison of “2 h” and “24 h” groups. In this work, differences were found in the SRY elements, that commonly present in genes determining cell fate and differentiation in animals. We found that there was substantially less SRY element-containing genes belonging to the “general stress” group down-regulated by chronic exposure when compared to acute exposure (60% the acute groups and only 28% in chronic group). Similarly, the “transcription factors” group of genes had only 20–25% of SRY elements in the chronic group and over 60% in acute group. The SRY element is commonly found in plant genes (Fig. 6), and despite the fact that the function of SRY elements in plants is not clear, it is possible that its function is conserved in plants and that acute stresses result in more severe changes in cell differentiation and cell fate decisions than does chronic IR stress.

The HNF transcription factors are involved in cell differentiation and cell proliferation and are commonly regulated by stress. This element is rarely found in plant genes, so it was difficult to make any comparison between “acute” and “chronic” groups (Fig. 6). The ABRE element was found to be more abundant in the “general stress” group of genes that was up-regulated by chronic radiation as opposed to those of acute exposure. This element was found in all genes in the chronic group and only in less than 20% of genes in the acute group (Fig. 6). The group of down-regulated genes also contained more ABRE elements in the chronic group, 14% versus the 0% found in acute group. This further supports our theory that chronic stress regulates more genes that are helping in plant adaptation, whereas the acute stress regulates the genes necessary for immediate survival. This assumption would suggest that the chronic stress would induce more genes belonging to the “general stress” category, which would therefore contain various regulatory elements such as ABRE, core, and DRE. We have profiled promoters of several “unknown” and “undetermined” genes and found much lower frequency of the occurrence of ABRE, DRE, Oct and core elements (data not shown). Acute stress induced more genes belonging to “transcription factors” (and probably signaling pathways) that

I. Kovalchuk et al. / Mutation Research 624 (2007) 101–113

contain various regulatory elements such as MYC, core, CdxA, SRY. Indeed, as it can be seen in Fig. 6, regulatory elements like ABRE, CORE, CdxA were more commonly found in ‘general stress’ group of genes induced by chronic exposure, whereas regulatory elements like MYC, core, CdxA and SRY were more common in the acute group of “transcription factor” genes. Further, as can be seen from Fig. 4, acute stress more actively changes the expression of transcription factors. 4. Conclusion Our study suggests that plants respond to acute radiation in a similar way as it responds to other stresses. This type of response is often directed to immediate repair of the damage, activation of pro-survival mechanisms, and perhaps inhibition of cell division/cell differentiation. In contrast, chronic stress leads to a totally different response that reflects in adaptive responses by regulating genes belonging to general stress and nucleic acid metabolism. The latter is of great importance, as plant adaptation is associated with chromatin modifications and changes in methylation pattern, both having a transgenerational nature. The main, general conclusion that this study suggests is that acutely exposed plants have to respond quick and hard to survive, whereas chronically exposed plants have to adjust and fine-tune their physiology. Acknowledgements We want to thank Edward Oakeley for his help in analysis of microchip data and Barbara Hohn, Franz Zemp, Alex Boyko and Scott Greer for critical comments on the manuscript. The NSERC and Alberta Ingenuity Grants are acknowledged for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.mrfmmm.2007.04.009. References [1] S.P. Jackson, Detecting, signalling and repairing DNA doublestrand 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 Studies, 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 (Pinus silvestris) stands post-Chernobyl, Sci. Total Environ. 157 (1994) 383–386.

111

[4] V.Y. Dashlers, I.D. Rashals, The influence of gamma or neutron radiation on the changes of plant productivity in populations of Arabidopdsis thaliana in eight generations, Arabidopsis Inform. Serv. 14 (1977). [5] J.P. Witherspoon, A.K. Corney, Differential and combined effects of beta, gamma and fast neutron irradiation of soybean seedlings, Radiat. Bot. 3 (1970) 125–133. ˇ [6] J. Paradiz, J. Skrk, B. Druˇskovic, Cytogenetic effects of ionizing radiation on meristem, Acta Pharm. 42 (1992) 397–401. [7] I.G. Mednic, P.D. Usmanov, Influence of gamma-rays on the number of initial cell in Arabidopsis thaliana, Arabidopsis Inform. Serv. 22 (1985) 65–70. [8] V.V. Schevchenko, L.I. Grinikh, Spectrum of chlorophyll deficient mutations induced by gamma-irradiation of Arabidopsis thaliana at different stages of development and scored with embryo test, Arabidopsis Inform. Serv. 18 (1981) 127–129. [9] I. Kovalchuk, O. Kovalchuk, A. Arkhipov, B. Hohn, Transgenic plants are sensitive bioindicators of nuclear pollution caused by the 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 exposure to ionizing radiation exhibit higher frequency of homologous recombination than acutely irradiated plants, Mutat. Res. 449 (2000) 47–56. [11] S. Ichikawa, C. Takahashi, Somatic mutation frequencies in the stamen hairs of stable and mutable clones of Tradescantia after acute gamma-ray treatments with small doses, Mutat. Res. 45 (1977) 195–204. [12] S. Ichikawa, Tradescantia stamen-hair system as an excellent botanical tester of mutagenicity: its responses to ionizing radiation and chemical mutagens, and some synergistic effects found, Mutat. Res. 270 (1992) 3–22. [13] V.I. Abramov, O.M. Fedorenko, V.A. Shevchenko, Genetic consequences of radioactive contamination for populations of Arabidopsis, Sci. Total Environ. 112 (1992) 19–28. [14] A.B. Syomov, S.N. Ptitsyna, S.A. Sergeeva, Analysis of DNA strand break induction and repair in plants from the vicinity of Chernobyl, Sci. Total Environ. 112 (1992) 1–8. [15] E.L. Kordium, P.G. Sidorenko, The results of the cytogenetic monitoring of the species of angiosperm plants growing in the area of the radionuclide contamination after the accident at the Chernobyl Atomic Electric Power Station, Tsitol. Genet. 31 (1997) 39–46. [16] E.I. Ziablitskaia, S.A. Geras’kin, A.A. Udalova, E.V. Spirin, An analysis of the genetic sequelae of the contamination of winter rye crops by the radioactive fallout from the Chernobyl Atomic Electric power station, Radiat. Biol. Radioecol. 36 (1996) 498–505. [17] I. Kovalchuk, V. Titov, B. Hohn, O. Kovalchuk, Transcriptome profiling reveals similarities and differences in plant responses to cadmium 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 and other 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. Magniette, S. Cuine, P. Auroy, P. Richaud, C. Forestier, J. Bourguignon, J.P. Renou, A. Vavasseur, N. Leonhardt, Genome-wide transcriptome profiling of the early cadmium response of Arabidopsis roots and 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,

112

[22]

[23]

[24] [25]

[26]

[27]

[28]

[29]

[30] [31]

[32]

[33]

[34]

[35]

[36]

[37]

I. Kovalchuk et al. / Mutation Research 624 (2007) 101–113 Transcriptomic footprints disclose specificity of reactive oxygen species signaling in Arabidopsis, Plant Physiol. 141 (2006) 436–445. R. Thilmony, W. Underwood, S.Y. He, Genome-wide transcriptional analysis of the Arabidopsis thaliana interaction with the plant pathogen Pseudomonas syringae pv. tomato DC3000 and the human pathogen Escherichia coli O157:H7, Plant J. 46 (2006) 34–53. P. Swoboda, S. Gal, B. Hohn, H. Puchta, Intrachromosomal homologous recombination in whole plants, EMBO J. 13 (1994) 484–489. A. Moiseev, V. Ivanov, Directory for Dosimetry and Radiation Hygiene, Atomizdat, Moscow, 1991, p. 220. N.I. Sanzharova, V.A. Kotik, A.N. Arkhipov, G.A. Sokolik, Iu.A. Ivanov, S.V. Fesenko, S.E. Levchuk, The quantitative parameters of the vertical migration of radionuclides in the soils in different types of meadows, Radiat. Biol. Radioecol. 36 (1996) 488– 497. R. Jefferson, Assaying chimeric genes in plants: the GUS gene fusion system, Plant Mol. Biol. Reporter 5 (1987) 387– 405. Y.E. Dubrova, V.N. Nesterov, N.G. Krouchinsky, V.A. Ostapenko, R. Neumann, D.L. Neil, A.J. Jeffreys, Human minisatellite mutation rate after the Chernobyl accident, Nature 380 (1996) 683–686. C. Mothersill, C. Seymour, Radiation-induced bystander and other non-targeted effects: novel intervention points in cancer therapy? Curr. Cancer Drug Targets 6 (2006) 447– 454. E.G. Wright, P.J. Coates, Untargeted effects of ionizing radiation: implications for radiation pathology, Mutat. Res. 597 (2006) 119–132. U. Wittstock, B.A. Halkier, Glucosinolate research in the Arabidopsis era, Trends Plant Sci. 7 (2002) 263–270. H. Duan, M.A. Schuler, Differential expression and evolution of the Arabidopsis CYP86A subfamily, Plant Physiol. 137 (2005) 1067–1081. 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 plants by cytochrome P450 inhibitors, Bioorg. Med. Chem. 13 (2005) 4491–4498. K. Sun, Y. Cui, B.A. Hauser, Environmental stress alters genes expression and induces ovule abortion: reactive oxygen species appear as ovules commit to abort, Planta 222 (2005) 632– 642. T. Ishikawa, Y. Morimoto, R. Madhusudhan, Y. Sawa, H. Shibata, Y. Yabuta, A. Nishizawa, S. Shigeoka, Acclimation to diverse environmental stresses caused by a suppression of cytosolic ascorbate peroxidase in tobacco BY-2 cells, Plant Cell Physiol. 46 (2005) 1264–1271. G.H. Badawi, N. Kawano, Y. Yamauchi, E. Shimada, R. Sasaki, A. Kubo, K. Tanaka, Over-expression of ascorbate peroxidase in tobacco chloroplasts enhances the tolerance to salt stress and water deficit, Physiol. Plant. 121 (2004) 231–238. I. Murgia, D. Tarantino, C. Vannini, M. Bracale, S. Carravieri, C. Soave, Arabidopsis thaliana plants overexpressing thylakoidal ascorbate peroxidase show increased resistance to Paraquatinduced photooxidative stress and to nitric oxide-induced cell death, Plant J. 38 (2004) 940–953. I. Thoma, C. Loeffler, A.K. Sinha, M. Gupta, M. Krischke, B. Steffan, T. Roitsch, M.J. Mueller, Cyclopentenone isoprostanes

[38]

[39]

[40]

[41]

[42]

[43] [44] [45]

[46] [47]

[48]

[49]

[50]

[51]

[52]

[53]

induced by reactive oxygen species trigger defense gene activation and phytoalexin accumulation in plants, Plant J. 34 (2003) 363–375. M.W. Bianchi, C. Roux, N. Vartanian, Drought regulation of GST8, encoding the Arabidopsis homologue of ParC/Nt107 glutathione transferase/peroxidase, Physiol. Plant 116 (2002) 96–105. A.P. Smith, S.D. Nourizadeh, W.A. Peer, J. Xu, A. Bandyopadhyay, A.S. Murphy, P.B. Goldsbrough, Arabidopsis AtGSTF2 is regulated by ethylene and auxin, and encodes a glutathioneS-transferase that interacts with flavonoids, Plant J. 36 (2003) 433–442. 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 auxin signaling pathways, is involved in salt stress response and lateral root development, Plant J. 44 (2005) 903–916. N.S. Mishra, R. Tuteja, N. Tuteja, Signaling through MAP kinase networks in plants, Arch. Biochem. Biophys. 452 (2006) 55– 68. Y.H. Cheong, H.S. Chang, R. Gupta, X. Wang, T. Zhu, S. Luan, Transcriptional profiling reveals novel interactions between wounding, pathogen, abiotic stress, and hormonal responses in Arabidopsis, Plant Physiol. 129 (2002) 661–677. K. Sax, The effect of ionizing radiation on plant growth, Am. J. Bot. 42 (1955) 360–364. J.E. Gunckel, The effects of ionizing radiation on plants: morphological effects quarterly, Rev. Biol. 32 (1957) 46–56. K. Daly, K.H. Thompson, Quantitative dose-response of growth and development in Arabidopsis thaliana exposed to chronic gamma-radiation, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 28 (1975) 61–66. G. Fox, Drought and the evolution of flowering time in desert annuals, Am. J. Bot. 77 (1990) 1508–1518. C. Martinez, E. Pons, G. Prats, J. Leon, Salicylic acid regulates flowering time and links defence responses and reproductive development, Plant J. 37 (2004) 209–217. Y. Sakuma, K. Maruyama, Y. Osakabe, F. Qin, M. Seki, K. Shinozaki, K. Yamaguchi-Shinozaki, Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in drought-responsive gene expression, Plant Cell 18 (2006) 1292– 1309. C. Temme, S. Zaessinger, S. Meyer, M. Simonelig, E. Wahle, A complex containing the CCR4 and CAF1 proteins is involved in mRNA deadenylation in Drosophila, EMBO J. 23 (2004) 2862–2871. K.W. Mulder, G.S. Winkler, H.T. Timmers, DNA damage and replication stress induced transcription of RNR genes is dependent on the Ccr4-Not complex, Nucleic Acids Res. 33 (2005) 6384–6392. 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 S phases is CCR4 dependent following ionizing radiation or replication stress in Saccharomyces cerevisiae, Eukaryot. Cell 3 (2004) 430–446. Y. Yu, Y. Teng, H. Liu, S.H. Reed, R. Waters, UV irradiation stimulates histone acetylation and chromatin remodeling at a repressed yeast locus, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 8650– 8655. M. Benhamed, C. Bertrand, C. Servet, D.X. Zhou, Arabidopsis GCN5, HD1, and TAF1/HAF2 interact to regulate histone acetylation required for light-responsive gene expression, Plant Cell (2006) [Epub ahead of print].

I. Kovalchuk et al. / Mutation Research 624 (2007) 101–113 [54] Y. Saintigny, F. Delacote, D. Boucher, D. Averbeck, B.S. Lopez, XRCC4 in G1 suppresses homologous recombination in S/G2, in G1 checkpoint-defective cells, Oncogene (2006) [Epub ahead of print]. [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-

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, Microarray analysis of genes that respond to gamma-radiation in Arabidopsis, J. Agric. Food Chem. 53 (2005) 1022–1030.