Present address: Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, PO Box 7080, SE-75007 Uppsala, Sweden.
Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
The multifunctional protein kinase CK2 is involved in several aspects of the DNA damage response (DDR) in mammals. To gain insight into the role of CK2 in plant genome maintenance, we studied the response to genotoxic agents of an Arabidopsis CK2 dominant-negative mutant (CK2mut plants). CK2mut plants were hypersensitive to a wide range of genotoxins that produce a variety of DNA lesions. However, they were able to activate the DDR after exposure to γ irradiation, as shown by accumulation of phosphorylated histone H2AX and up-regulation of sets of radio-modulated genes. Moreover, functional assays showed that mutant plants quickly repair the DNA damage produced by genotoxins, and that they exhibit preferential use of non-conservative mechanisms, which may explain plant lethality. The chromatin of CK2mut plants was more sensitive to digestion with micrococcal nuclease, suggesting compaction changes that agreed with the transcriptional changes detected for a number of genes involved in chromatin structure. Furthermore, CK2mut plants were prone to transcriptional gene silencing release upon genotoxic stress. Our results suggest that CK2 is required in the maintenance and control of genomic stability and chromatin structure in plants, and that this process affects several functions, including the DNA damage response and DNA repair.
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The integrity of DNA is continuously challenged within the cell. To counteract the severe biological consequences of DNA damage, an intricate network of genome surveillance mechanisms, often referred to as DNA damage responses (DDRs), has evolved. Most of the DDR components identified in animals and yeasts have counterparts in plants (Britt, 1996, 1999; Bray and West, 2005; Kimura and Sakaguchi, 2006). However, DNA repair pathways in animals include some components for which homologues have not been found in plants, and some DDR regulators are unique to plants (Britt, 1999; Kimura and Sakaguchi, 2006; Yoshiyama et al., 2009).
The initial stages of the DDR in mammals are governed by a pair of closely related protein kinases, termed ataxia telangiectasia mutated (ATM) and ATM- and RAD3-related (ATR). ATM and ATR function as sensors of DNA damage, and control the phosphorylation of histone H2AX in regions close to damaged chromatin (Kinner et al., 2008; Mah et al., 2010). Phosphorylated H2AX (γ-H2AX) mediates the formation of DNA damage foci, which are large aggregates of proteins and repair factors that surround the lesion sites (Riches et al., 2008). Plants possess ATM and ATR orthologues (Garcia et al., 2003; Culligan et al., 2004). ATM senses double-strand breaks (DSBs), triggering a transcriptional response to ionizing radiation (IR) (Garcia et al., 2003), whereas ATR senses repair intermediates or stalled replication forks and has a prominent role in the UV-induced response (Culligan et al., 2004; Yoshiyama et al., 2009; Furukawa et al., 2010). Both ATR and ATM are involved in IR-induced phosphorylation of H2AX in Arabidopsis, although ATM is responsible for the majority of focus formation in M-phase cells (Friesner et al., 2005). IR-induced DSBs are among the most harmful lesions in DNA, and must therefore be eliminated before chromosome segregation. In eukaryotes, DSBs can be repaired by two major pathways: homologous recombination (HR) and non-homologous DNA end joining (NHEJ). HR is a high-fidelity mechanism that uses unbroken, homologous sequences to template repair of DSBs. It is mostly used to repair DSB breaks due to DNA replication, using sister chromatid information (Orel et al., 2003). In contrast, NHEJ does not require significant sequence homology for DSB repair, and is a prominent mechanism in eukaryotes (Lieber, 2010). In plants, four major non-homologous recombination pathways have been described: (i) canonical non-homologous end joining (C-NHEJ), which is Ku-dependent, (ii) alternative end joining (A-EJ or A-NHEJ), which is more error-prone and uses microhomologies for recombination (also called MMEJ), (iii) the back-up pathway (B-NHEJ), which is Ku-independent and involves Parp1, Xrcc1 and DNA ligase III, and (iv) an unidentified pathway that confers severe genomic instability (Charbonnel et al., 2011). A hierarchical organization of these pathways during post-S phase has been proposed in Arabidopsis (Charbonnel et al., 2011).
DNA damage responses are tightly linked to chromatin structure. In plants, mutations in replication-coupled chromatin assembly factor or chromatin assembly factor 1 (CAF-1) result in sensitivity to DNA damage, de-repression of transcriptional silencing, and genome instability (Takeda et al., 2004; Kirik et al., 2006; Schonrock et al., 2006). Chromatin is also a major factor in the regulation of the HR pathway, as exemplified by different Arabidopsis mutants, such as fas1-4, which is defective in the p150 subunit of CAF-1, mim, which lacks a chromatin structural component related to the structural maintenance of chromosomes (SMC) family (Mengiste et al., 1999; Kirik et al., 2006), and bru1, which encodes a nuclear protein of unknown function (Takeda et al., 2004).
Here we have used an inducible dominant-negative allele of CK2 to investigate the role of this protein kinase in damage tolerance and DNA repair in the higher plant Arabidopsis. We demonstrate that lack of CK2 activity confers hypersensitivity to a variety of genotoxic agents, although CK2-defective plants displayed normal levels of IR-induced histone H2AX phosphorylation and are able to activate a transcriptional response. Our results suggest that mutant plants show preferential use of non-conservative pathways for DNA repair. Moreover, changes in chromatin structure and de-repression of transcriptional gene silencing suggest that CK2 is required in the maintenance of chromatin structure, sustaining the viability of plant cells despite the deleterious effects of genotoxic agents.
Arabidopsis plants depleted of CK2 activity are hypersensitive to genotoxins
In previous studies, we demonstrated that an inducible dominant-negative allele of CK2 (CK2mut) could be successfully used to deplete CK2 activity in Arabidopsis plants (Moreno-Romero et al., 2008; Marques-Bueno et al., 2011). Here we have used the same mutant to study the involvement of CK2 in plant DDRs. We performed transient inductions of the transgene (typically treatment of 5-day-old seedlings with dexamethasone for 48 h), followed by genotoxic treatment on the 7th day. Unless otherwise indicated, uninduced CK2mut plants were used as controls. Our results show that mutant seedlings are hypersensitive to IR at 100 Gy (Figure 1a), exhibiting anthocyanin accumulation, cotyledon necrosis and developmental arrest. The effects are specifically due to accumulation of CK2mut protein, as dexamethasone treatment had no effects on γ-irradiated wild-type or atm plants. The Arabidopsis atm mutant is a well-known IR-hypersensitive mutant that is defective in the DDR response. Moreover, CK2mut plants were hypersensitive to IR when dexamethasone treatment was performed before or after γ irradiation (Figure 1b). As Arabidopsis mutants affected in DNA repair processes show defects in root growth under genotoxic stress (Garcia et al., 2003; Culligan et al., 2006), we measured daily root growth in post-IR CK2mut seedlings. Root growth was permanently arrested upon 80 Gy treatment in CK2mut seedlings (Figure 1c, left; note that CK2mut roots were initially shorter, as previously described by Moreno-Romero et al., 2008), whereas the arrest was transient in Arabidopsis wild-type. Moreover, root viability appeared highly compromised in CK2mut plants (Figure 1c, right).
We have recently shown that depletion of CK2 activity alters polar auxin transport (Marques-Bueno et al., 2011). In order to ascertain whether the IR hypersensitivity was an indirect effect of auxin transport impairment, we treated CK2mut and control seedlings with either indole-3-acetic acid (IAA) or N-1-naphtylphtalamic acid (NPA) (an inhibitor of polar auxin transport) prior to IR exposure. Quantification of plant fresh weight in post-IR 20-day-old plants showed that the IR hypersensitivity of CK2mut seedlings was not reversed by exogenous IAA (Figure S1), in contrast to other CK2mut phenotypes previously reported by Marques-Bueno et al. (2011). Moreover, NPA slightly affected the growth of post-irradiated control plants, but did not have deleterious effects. Altogether, these data led us to conclude that the hypersensitivity phenotype shown by CK2mut plants is not an indirect consequence of impaired auxin transport.
We also tested the sensitivity of CK2mut plantlets to UV-C and methyl methanesulfonate (MMS). CK2mut plants were hypersensitive to UV-C radiation, showing significant growth inhibition at 30 000 J m−2 (Figure 1d), and to MMS over a range of 25–100 ppm (Figure 1e).
Taken together, these results strongly suggest that CK2 is required to successfully recover from different genotoxic treatments in Arabidopsis. The genotoxins tested are known to produce different kinds of lesions into DNA, and thus CK2 may act upstream of different signalling pathways necessary for DNA repair. Alternatively, CK2 may be required at multiple points or may play a more general role in plant survival.
For the subsequent studies, we focused on IR-induced responses using γ irradiation. IR-induced responses have been well characterized in Arabidopsis wild-type, facilitating their study in mutant genotypes. Moreover, γ irradiation has a DSB production ratio (number of DSBs/base pair versus dose) that is similar in all eukaryotes (Su, 2006).
CK2 expression and activity do not significantly change after exposure to IR
The CK2α and β subunits are encoded by two small multigene families. The Arabidopsis nuclear genome contains four genes for the catalytic subunit (α) and four genes for the regulatory subunit (β) (Salinas et al., 2006). The IR-induced expression changes of CK2-encoding genes were very small, never reaching twofold, whereas TSO2 (encoding the small subunit of ribonucleotide reductase, which was used as a positive control) was highly over-expressed under the same conditions (Figure S2a). We also analyzed CK2 expression data from the AtGenExpress project, which compiles Arabidopsis data from ATH1 GeneChip arrays (Kilian et al., 2007). CK2α and β transcript levels showed no significant changes in Arabidopsis plants (18-day-old plantlets, Col-0 ecotype) treated with 1.5 μg ml−1 bleomycin (a radiomimetic) plus 22 μg ml−1 mitomycin C (a cross-linking agent) (Figure S2b). Although the plant ages and genotoxins used in the array experiments were different from those employed in our work, both set of data support the idea that CK2 is not transcriptionally regulated in response to DNA-damaging agents. This is consistent with data showing that mammalian CK2 activity is regulated mainly at a post-translational level (Filhol and Cochet, 2009). However, no significant changes of CK2 activity were found in γ-irradiated Arabidopsis wild-type plantlets (Figure S2c).
IR-induced global changes of gene expression in CK2mut seedlings
Plants subjected to genotoxic stress become impaired in a wide number of cellular functions. Particularly well known is the IR-induced transcriptional burst in Arabidopsis plants, affecting a large number of genes (Chen et al., 2003; Nagata et al., 2005; Culligan et al., 2006; Kim et al., 2007; Ricaud et al., 2007). We analyzed transcript profiles of γ-irradiated mutant plants (100 Gy) using Affymetrix ATH1 chips. The study was performed at 1.5 h post-IR, as transcript radio-modulation in Arabidopsis occurs as an early wave after IR and lasts approximately 3 h, with only 10% of the genes still showing changes 5 h post-IR (Ricaud et al., 2007). The experimental design and comparative analysis performed are summarized in Figure 2(a). Pairwise comparisons revealed different numbers of affected genes depending on the variable analyzed (Figure 2a, bottom). For instance, induction of the transgene (pairwise comparison number 2, ‘CK2mut effect’) produced the widest changes, affecting 6614 sequences (P <0.001) out of the 22 746 sequences present in the array (after subtracting the changes obtained in plants transformed with the empty vector, shown in comparison number 1). IR-induced transcriptional changes in control plants (pairwise comparison number 3, ‘gamma effect’) correlated well with those reported in the literature for Arabidopsis wild-type plants (Table S1a).
Genes showing IR-induced transcriptional changes in control and mutant seedlings were grouped into GO functional categories, depicted in Figure 2(b) (‘gamma effect’ and ‘CK2mut + gamma effect’, respectively). The number of genes within each category is shown in Table S1a. There are four shared categories, but the number of genes within them and/or their statistical significance differ between the two conditions. Ninety genes were present in both datasets (‘CK2mut + gamma effect’ and ‘gamma effect’), 89 showing positive correlation (59 up-regulated and 30 down-regulated in both cases) and one showing negative correlation (up-regulated in ‘CK2mut + gamma effect’ and down-regulated in ‘gamma effect’) (Figure 2c, left, and Table S1b). The IR-induced fold changes in expression were lower in mutant than control plants (see the slope of the correlation plot in Figure 2c, right), and this was confirmed by RT-PCR for some of these genes (Figure S3). A time-course study showed that gene under-expression in the mutant was due to weaker induction not induction delay (Figure 2d).
To gain insight into the nature of the IR-induced transcriptional response, we further analyzed the genes specifically involved in DNA repair processes. One hundred and fifty genes have a known or predicted function in DNA repair (http://www.uea.ac.uk/~b270/repair.htm). Some are radio-modulated (rapidly IR-induced) (Schonrock et al., 2006; Dohmann et al., 2008), although the majority are not clearly induced after DNA damage (http://bar.utoronto.ca/ or http://www.weigelworld.org/resources/microarray/AtGenExpress). DNA repair genes with significant changes in our experimental conditions (anova, P value <0.001) were classified into DNA repair pathways. Many of those genes were up- or down-regulated in non-irradiated CK2mut seedlings, although the fold changes were rather modest (Figure 2e and Table S1c). In particular, several of the genes involved in HR appeared to be transcriptionally repressed, but no major changes were found in the genes of the NHEJ pathways. Analysis of array data from other authors (Menges et al., 2005) showed that genes involved in HR are cell cycle-regulated, with peaks of expression at S phase, whereas genes involved in NHEJ pathways show steady-state expression levels during the cell cycle (Figure S4a). We have previously demonstrated that CK2mut gene expression leads to cell-cycle arrest at G1/S (Moreno-Romero et al., 2008), which agrees with the expression profile of cell cycle-regulated genes in mutant seedlings (array data, Figure S4b). Therefore, transcriptional under-expression of the HR pathway in mutant plants may be due to G1/S cell-cycle arrest. In spite of this, our array data also show that radio-modulated genes involved in the HR pathway (such as RAD51 or BRCA1) were IR-induced in mutant plants (Figure 2e), indicating that the pathway may be activated in a cell cycle-independent manner under stress conditions.
CK2mut seedlings show high DNA repair proficiency
The DNA repair proficiency of mutant seedlings was investigated by comet assays. The amount of DNA in the tail (Figure 3a) was measured before and after treatments with two different genotoxins (bleomycin or IR), using two different protocols (the N/N protocol that detects DSBs, and the A/A protocol that detects DSBs, SSBs and the majority of labile sites; Menke et al., 2001). Surprisingly, comet assays performed on bleomycin-treated plants with the N/N protocol showed that DSBs were more rapidly repaired in mutant than in control plants (Figures 3b and S5a). Similar results were obtained using the A/A protocol in combination with γ irradiation (100 Gy): the tail of the comets disappeared faster in mutant plants (Figure 3c). As a negative control, we used the IR-hypersensitive atm mutant, which did not show enhanced DNA repair rates in our assays (Figure S5a,b). These results suggest that mutant plants are more proficient in repairing DSBs and other secondary lesions produced by IR or bleomycin, despite their hypersensitivity to these agents.
CK2-defective plants show decreased homologous recombination
To analyze the efficiency of DSB repair by homologous recombination (HR), we used two recombinogenic substrates present in Arabidopsis transgenic lines generated by H. Puchta (Botanisches Institut, Universität Karlsruhe, Germany). HR events were detected by restoration of β-glucuronidase (GUS) activity, which was measured as a blue precipitate (Orel et al., 2003). CK2 activity was inhibited prior the assay by incubation of plants with tetrabromobenzotriazol (TBB) (Shugar, 1994), a specific inhibitor that does not interfere with the activity of other Arabidopsis kinases (Espunya et al., 1999). TBB-treated seedlings exhibited fewer blue sectors than control plants in all the conditions tested (Figure 3d), indicating lower HR frequency. However, the number of blue sectors significantly increased upon IR (blue spots were seen 24 h after IR), both in control and TBB-treated seedlings, suggesting that CK2 is not required to activate the HR pathway. In order to check whether the lower HR efficiency of TBB-treated plants was due to an indirect effect of cell-cycle arrest produced by CK2 inhibition, we assessed the effect of aphidicolin, a drug that blocks the cell cycle at G1/S, on HR proficiency. Figure 3(e) shows that, in non-irradiated seedlings, the number of HR events was similarly decreased in the presence of either aphidicolin or TBB (blue spots were seen 72 h after IR). Moreover, γ irradiation induced significant higher HR activation in aphidicolin-treated plants than in TBB-treated plants (1.85-fold versus 1.47-fold, respectively), and the simultaneous presence of TBB and aphidicolin did not have an additive effect on HR activation. To assess the long-term effects of aphidicolin on plants’ sensitivity to IR, plants were irradiated with 100 Gy and fresh weights were measured 13 days after IR. Figure 3(f) shows that IR affects growth significantly more in CK2-defective plants than in aphidicolin-treated plants. Moreover, aphidicolin-treated plants were able to develop true leaves, whereas CK2-defective plants were developmentally arrested. As previously demonstrated, a 100 Gy dose is lethal for CK2mut plants, but not for aphidicolin-treated plants. We conclude that, although cell-cycle arrest may contribute to the weaker HR activity in non-irradiated CK2-defective seedlings, additional mechanisms may be responsible for the impaired DDR in the same plants.
IR-induced histone H2AX phosphorylation and cyclin CYCB1;1 expression are unaffected in CK2mut plants
One of the first molecular events induced by the DDR is phosphorylation of histone H2AX. To determine whether this mechanism was activated in CK2mut plants, protein extracts from γ-irradiated (100 Gy) seedlings were prepared at 20 min after IR, and immunoblotted with an antiserum specific to the phosphorylated form of H2AX (γ-H2AX). We detected a band of the expected molecular weight, the intensity of which was similar in mutant and control plants (Figure 4a). This finding was corroborated by performing a time-course analysis of γ-H2AX accumulation: both mutant and control plants showed the maximum band intensity at 20 min after IR, rapidly decreasing afterwards (Figure 4b).
Another molecular marker of DDR activation is up-regulation of mitotic cyclin CYCB1;1 (Culligan et al., 2006). CYCB1;1 accumulation was monitored during 3 days post-IR (100 Gy), using the translational fusion CYCB1;1::GFP introduced into the CK2mut background. GFP fluorescence was similar in γ-irradiated mutant and control plants (Figure 4c), although the initial CYCB1;1 levels were lower in the mutant, as reported previously (Moreno-Romero et al., 2008).
These data indicate no significant changes in the initial responses of CK2mut plants to IR-induced DNA damage, confirming that their phenotype of hypersensitivity is not due to defects in the activation of signalling pathways.
Chromatin structure in CK2mut plants
Due to the close relationship between chromatin structure and the DNA damage response, we assessed the sensitivity of the chromatin of CK2mut plants to micrococcal nuclease (MNase). MNase is able to produce nicks in the inter-nucleosomal DNA, and its efficiency depends on the degree of chromatin compaction. Figure 5(a) shows that the chromatin from CK2mut plants was more sensitive to MNase digestion than the chromatin from control plants (Figure 5a). This was confirmed by performing a time-course analysis with 2.5 U ml−1 of nuclease at 4°C (Figure 5b) or 37°C (Figure S6a). In all the conditions tested, nuclei from CK2mut plants were digested faster, leading to accumulation of DNA bands of lower molecular weight (Figure S6b). Several laboratories have demonstrated a genetic link between chromatin structure, DNA repair and transcriptional gene silencing (Takeda et al., 2004; Elmayan et al., 2005). The array data from CK2mut seedlings showed significant transcriptional changes in genes involved in chromatin structure, such as histone modification, heterochromatin formation or nucleosome assembly (Figure S7a). Moreover, determination of transcript levels of FAS1 and FAS2 (encoding two subunits of the CAF-1 complex) and of MIM by RT-PCR showed that they were under-expressed in mutant seedlings, whereas those of MSI1 (the third subunit of CAF-1) did not show significant changes (Figure 5c). It has been reported that loss-of-function fas1, fas2 and mim mutants are hypersensitive to MMS, and that fas1 and fas2 mutations activate the transcriptionally silent information (TSI) element (Mengiste et al., 1999; Takeda et al., 2004). TSI repeats, concentrated in pericentromeric chromosomal regions, are heavily methylated and silenced in wild-type Arabidopsis, and their expression is activated in some mutants affected in epigenetic regulation (Steimer et al., 2000). In contrast, CK2-defective plants show normal basal TSI transcript levels. Upon IR, TSI levels increased 13.5-fold in CK2mut plants and only 2.8-fold in control plants (Figure 5d). In order to assess whether the IR-induced TSI over-expression in CK2mut plants was an indirect effect of the altered auxin responses reported for this mutant (Marques-Bueno et al., 2011), we measured TSI levels in post-IR plants previously incubated with either the synthetic auxin analogue 1-naphthaleneacetic acid (NAA) or the auxin transport inhibitor NPA. Figure 5(e) shows that neither compound affects TSI levels in wild-type plants, indicating that impairment of auxin transport is likely not responsible for the TSI release of silencing. Interestingly, incubation with NPA increases TSI over-expression and IR sensitivity in CK2mut plants (Figures 5e and S1, respectively).
In addition, microarray data showed that gene silencing in either telomeric regions (data were analyzed as in Schonrock et al., 2006, for telomeric genes present in the array), pericentromeric regions of chromosomes 2 and 4 (Nakahigashi et al., 2005), or transposable-related elements, was maintained in CK2mut plants (Figure S7b,c,d, respectively). We could not analyze gene expression in heterochromatic regions in γ-irradiated CK2mut plants, because the array data were obtained at 1.5 h post-IR, which is too short a time to detect such changes. However, the above data enabled us to conclude that CK2mut plants have an altered, less compacted chromatin structure, and that these plants show de-repression of gene silencing in the presence of stressors.
Studies in yeast and mammals suggest that the protein kinase CK2 is involved in the DNA damage response. This was supported by experimental data showing that CK2 mutants or inhibition of CK2 activity caused hypersensitivity to DNA-damaging agents. Moreover, key proteins of the DDR machinery have been shown to be phosphorylated by CK2 (Krohn et al., 2003; Koch et al., 2004; Loizou et al., 2004; Cheung et al., 2005; Yamane and Kinsella, 2005). The Arabidopsis mutant used in this work (CK2mut plants) exhibits hypersensitivity to a broad range of genotoxins, such as IR, UV-C, bleomycin and MMS. Moreover, like the much-studied Arabidopsis atm mutant (Garcia et al., 2003; Culligan et al., 2006), CK2mut root tips do not recover from exposure to 100 Gy IR, and, instead, terminally differentiate into root hair-producing organs. Interestingly, hypersensitivity to IR was detected when CK2 activity was depleted either before or after irradiation, suggesting that CK2 participates in long-term DNA damage responses and/or survival.
Unexpectedly, comet assays showed that CK2mut plants exhibited enhanced proficiency in repairing IR- and bleomycin-induced DSB lesions. DSB repair involves two different pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). The efficiency of the HR pathway was checked by using two recombinant substrates, DGU.US and DU.GUS, which exploit restoration of GUS marker gene by two alternative mechanisms: Rad51-independent (for DGU.US) and Rad51-dependent (for DU.GUS). CK2-defective plants showed a lower number of HR events with either substrate in both irradiated and non-irradiated plants. This supports the idea that HR is less active in CK2-defective plants, and that the rapid DSB repair activity measured by comet assays is due to enhanced NHEJ mechanisms. Our experiments with aphidicolin demonstrated that cell-cycle blockage at G1/S has a clear negative impact on HR activation, but this is not sufficient to explain the lower number of HR events in post-irradiated CK2-defective seedlings. According to the hierarchical action of DSB repair pathways proposed by Charbonnel et al. (2011), HR is a late-onset pathway that repairs DSBs that were not processed by NHEJ. As CK2mut plants show very rapid DNA repair kinetics, a possible explanation is that most breaks are repaired by NHEJ pathways, which involves the use of non-conservative mechanisms and can lead to loss of genetic material and/or accumulation of errors that may be deleterious. In particular, the still unidentified fourth pathway postulated by Charbonnel et al. (2011) that confers severe genomic instability might be favoured in CK2mut plants.
ATM-dependent phosphorylation of histone H2AX, one of the earlier responses to DSB induction, is conserved in plants (Friesner et al., 2005). The kinetic of H2AX phosphorylation/dephosphorylation in IR-treated CK2mut plants strongly suggests that both sensing of the stimulus and activation of the early steps in the signalling cascade are not affected by loss of CK2 activity. This is concordant with the observation that radio-modulated genes are induced at the appropriate times in the mutant, although to a lesser extent than in wild-type plants. According to some authors, inhibition of CK2 activity in mammals decreases the levels of γ-H2AX and the number of repair foci (Ayoub et al., 2008). However, other authors have recently reported that CK2 inhibition only delays γ-H2AX foci removal, reducing survival of irradiated mammal cells (Zwicker et al., 2011). In either case, CK2 in mammals appears to be involved in modulating γ-H2AX levels, whereas our results suggest that this is not the case in Arabidopsis. In mammals, CK2-driven phosphorylation of MDC1 is essential for accumulation and retention of the MRN complex and contributes to the propagation of γ-H2AX (van Attikum and Gasser, 2009). Although homologous genes for the MRN complex have been cloned in Arabidopsis and shown to play an important role in DNA repair and meiotic recombination (Waterworth et al., 2007), no plant homologues for MDC1 have been identified so far in plants. Therefore, animals and plants may differ mechanistically with regard to this process.
Chromatin dynamics is linked to DNA repair: minutes after damage, important changes in chromatin compaction occur, which are facilitated by histone modifications and chromatin remodelling (van Attikum and Gasser, 2005; Groth et al., 2007). In plants, many of the Arabidopsis mutants exhibiting hypersensitivity to genotoxic treatments are also affected in chromatin structure, such as fas1 and fas2 (Kirik et al., 2006), bru1 (Takeda et al., 2004), mre11 (Bundock and Hooykaas, 2002), mim (Mengiste et al., 1999), and npr1 and npr2 (Zhu et al., 2006). Some of these mutants also show de-repression of heterochromatic genes (Elmayan et al., 2005). The chromatin of CK2-defective plants was hypersensitive to MNase digestion, indicating either less compaction or structural disorganization. Moreover, the expression of genes such as FAS1 or MIM (which are involved in chromatin remodelling) was decreased, supporting the idea that CK2 activity regulates components directly involved in chromatin structure. However, transcriptional gene silencing was maintained in the heterochromatic structures analyzed (TSI, transposons and pericentromeric/telomeric regions), as late as 2 days after dexamethasone treatment, but, interestingly, TSI transcript levels increased dramatically when CK2mut seedlings were subjected to genotoxic stress. In this sense, CK2mut plants behaved similarly to npr1 and npr2 mutants, which show normal basal TSI levels but over-expression of this locus after genotoxic treatments. NPR1 and NPR2 encode histone chaperones that act in the maintenance of dynamic chromatin (Zhu et al., 2006).
In conclusion, our results show that CK2-defective Arabidopsis plants are hypersensitive to a wide range of genotoxic stress, suggesting that, as occurs in mammals, CK2 modulates the DDR in plants. CK2-defective plants are able to activate the DDR machinery, but preferentially use non-conservative mechanisms for DNA repair, which may explain plant lethality. The enhanced activity of NHEJ pathways may be facilitated in these plants because, in contrast to the HR pathway, expression of genes involved in NHEJ pathways is not cell cycle-dependent, and thus is unaffected by depletion of CK2 activity. Moreover, DNA damage sites may be more accessible to repair factors in CK2mut plants due to their less compacted chromatin, contributing to and amplifying the rapid use of NHEJ pathways instead of HR. In this scenario, CK2, by virtue of its dual activity as a regulator of chromatin remodelling and cell-cycle progression, plays a role in protecting cells from IR by avoiding extensive use of error-prone DNA repair mechanisms that, although quickly repairing the DNA, have deleterious effects.
Growth of plants, in vitro germination and culture, and generation of CK2mut and CYCB1;1::GFP x CK2mut transgenic plants have been described by Moreno-Romero et al. (2008). Seeds containing the recombinogenic substrates DGU.US and DU.GUS were kindly provided by Dr H. Putcha (Botanisches Institut, Universität Karlsruhe, Germany). Expression of the CK2mut transgene was induced with 1 μm dexamethasone dissolved in ethanol. Root lengths were measured on scanned plant images using a GS-700 imaging densitometer (Bio-Rad, Hercules, CA, USA) and imagej software (http://rsb.info.nih.gov/ij). Viability of root tip cells was tested by incubation with Evans blue (0.25% w/v) (Sigma-Aldrich, St Louis, MO, USA) for 1 h as described by Gaff and Okong’o-Ogola (1971). CYCB1;1::GFP expression was monitored with a DMRB microscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a Leica DC200 digital camera. Quantification of GFP fluorescence was performed using leica Q500MC software, in non-saturating conditions. IAA, NAA and NPA (Sigma-Aldrich) were dissolved in ethanol at the given concentrations and added to Petri dishes. Aphidicolin (Sigma-Aldrich) was dissolved in dimethylsulfoxide and used at 10 μg ml−1 for the indicated times.
Unless otherwise indicated, genotoxic treatments were performed on 7-day-old seedlings grown on MS plates or liquid medium. Ionizing radiation (IR) was provided by a 137caesium source generated by an IBL 437C irradiator (CIS Bio International, Bagnols/Ceze, France) at a dose rate of 5.7–5.5 Gy min−1. UV-C radiation was provided by a Stratalinker UV crosslinker (Stratagene, Agilent Technologies, Inc., Santa Clara, CA, USA). Bleomycin treatments were carried out for 1 h on 7-day-old seedlings grown on plates. MMS treatments were carried out for 7 days on 6-day-old seedlings grown in MS liquid medium and previously incubated with dexamethasone or ethanol for 24 h.
RNA extraction and quantitative RT-PCR assays were performed as described by Moreno-Romero et al. (2008). For semi-quantitative RT-PCR, the linear range of PCR product synthesis was established for each primer pair, and the number of cycles was chosen accordingly. Primer sequences and annealing temperature are shown in Table S2.
Hybridization and analysis of ATH1 Affymetrix arrays
Total RNA was isolated from frozen tissue samples using Trizol (Invitrogen, Carlsbad, CA, USA), and later purified using Qiagen RNeasy Plant Mini Kit columns (Qiagen, Valencia, CA, USA). Three independent RNA preparations were made from pooled samples for each of the conditions and lines. Microarray hybridizations (GeneChip® ATH1 from Affymetrix (Affymetrix, Inc., Santa Clara, CA, USA), with 22 810 sequences) were performed by the genomic facilities of Progenika Biopharma (Derio, Spain) using the methodology and equipment recommended by Affymetrix Inc. The complete array data for plants transformed with the empty vector (with and without dexamethasone) have been submitted to the Nottingham Arabidopsis Stock Centre database (reference NASCARRAYS-642; http://affymetrix.arabidopsis.info/).
Data analysis, including statistical analysis (anova, P value <0.001), fold change values, plot representations, clustering and Venn diagrams, was performed using rosetta resolver software (Rosetta Biosoftware, Seattle, WA, USA). Gene classification into functional categories was performed using BiNGO gene enrichment analysis (Maere et al., 2005). Microarray expression data of CK2 subunits were obtained from the AtGenExpress project (http://www.arabidopsis.org), data for the met1 mutant were obtained from Mathieu et al. (2007), and the list of transposable elements was obtained from Slotkin et al. (2009).
CK2 activity assay, extraction of histones and immunoblotting
CK2 kinase activity assays were performed as described by Moreno-Romero et al. (2008). The assays were done in triplicate, and background signals (values obtained after adding 1 μm TBB to a reaction test) were subtracted. Extraction of histones and immunoblotting were performed as described by Friesner et al. (2005).
Comet assays were performed as described by Menke et al. (2001), with some modifications. Briefly, frozen material was chopped with a fresh razor blade on ice and under dim light in PBS containing 10 mm EDTA. The suspensions of nuclei were filtered through 80 μm mesh Sefar Nytal (Sefar AG, Heiden Switzerland) and mixed with low-melting-point agarose (Bio-Rad) at 37°C, to a final concentration of 0.38% w/v (alkali, A/A protocol) or 0.19% w/v (neutral, N/N protocol). Drops of 70 μl were loaded onto microscope slides pre-coated with 1% agarose, and covered with 20 × 20 mm cover slips, then incubated on ice for 5 min. In the A/A protocol, the slides were incubated for 5 min in high-alkali buffer (0.3 m NaOH, 5 mm EDTA, pH 13.5), prior to electrophoresis at 4°C in the same buffer (0.7 V cm−1), followed by a short neutralization step of 5 min in 100 mm Tris/HCl, pH 7.5. In the N/N protocol, the slides were incubated for 1 h in high-salt buffer (2.5 m NaCl2, 10 mm Tris/HCl, pH 7.5, 100 mm EDTA) on ice, followed by an equilibration step in 1× TBE (90 mm Tris/borate, 2 mm EDTA, pH 8.4) (3 × 5 min) prior to electrophoresis (1 V cm−1) at room temperature in TBE buffer. The slides were then dehydrated in 75% v/v ethanol (5 min), followed by 96% v/v ethanol (5 min), and air-dried. Dry agarose gels were rehydrated for 5 min in water and stained with 4′,6-diamidino-2-phenylindole (DAPI) (1–5 μg ml−1). Images were captured using a Leica DRMB fluorescence microscope and quantified with cometscore software, version 1.5 (Tritek Corp., Sumerduck, VA, USA). At least 100 nuclei were scored from two different comet gels for each experiment.
Fourteen-day-old DGU.US or DU.GUS plantlets were treated with either 10 μm TBB for 16 h or 10 μg ml−1 aphidicolin, or both, for 24 h in liquid medium, and then γ-irradiated with 100 Gy and returned to the growth chamber for the indicated times in each experiment. Aphidicolin was maintained in the growth medium for the whole time, while TBB was removed after IR. The frequency of HR events was visualized as the number of blue tissue sectors after GUS histochemical staining. GUS activity was determined using a β-glucuronidase reporter gene staining kit (Sigma-Aldrich). Seedlings were mounted with 50% v/v glycerol and observed with a Leica DMRB microscope.
Micrococcal nuclease (MNase) assay
Nuclei from 7-day-old CK2mut plantlets (with or without dexamethasone) were isolated by grinding plant material (2 g) in liquid nitrogen, and then resuspended in 0.25 m sucrose, 60 mm KCl, 15 mm MgCl2, 1 mm CaCl2, 15 mm PIPES, pH 6.8, 0.8% Triton X-100 and 1 mm PMSF. The homogenate was centrifuged for 20 min at 10 000 g, and the pellet resuspended in 200–500 μl of 0.25 m sucrose, 10 mm Tris/HCl pH 8.0, 10 mm MgCl2, 1% v/v Triton X-100, 5 mm 2-mercaptoethanol and 1 mm PMSF. The suspension was placed on the top of an Eppendorf tube containing 1.7 m sucrose, 10 mm Tris/HCl, pH 8.0, 10 mm MgCl2, 0.5% v/v Triton X-100, 5 mmβ-mercaptoethanol and 1 mm PMSF, and centrifuged at 4°C for 1 h at 12 000 g. The resulting pellet was resuspended in MNase buffer (0.3 m sucrose, 20 mm Tris/HCl, pH 7.5, 3 mm CaCl2), and the DNA concentration was determined by measuring the absorbance at 260 nm. Equal amounts of nuclei were digested, and the reaction was stopped with 50 mm EDTA and 1% SDS. Samples were incubated with proteinase K overnight at 37°C, electrophoresed on agarose gels and visualized by gel staining with ethidium bromide.
We thank Dr H. Puchta (Botanisches Institut, Universität Karlsruhe, Germany) for providing the DGU.US and DU.US reporter lines. This work was supported by grants BFU2007-60569, BFU2010-15090 and Consolider Ingenio 2010 CSD2007-00036 from the Ministerio de Educación y Ciencia, (Spain) and grants 2005SGR-00112 and 2009SGR-795 from the Generalitat de Catalunya, Catalunya, Spain. J.M.-R. and L.A. were recipients of fellowships from the Universitat Autònoma de Barcelona and the Ministerio de Educación y Ciencia (Spain), respectively. We are indebted to the Arabidopsis Information Resource (TAIR) (http://arabidopsis.org) as an invaluable source of data, and to the Servei de Microscopia, Unitat Tècnica de Protecció Radioactiva and the Laboratori d’Anàlisi i Fotodocumentació (Universitat Autònoma de Barcelona, Spain) for technical support.