Abstract:
Described is the use of SMR5, possibly in combination with SMR4 and/or SMR7, to modulate ROS and oxidative stress response in plants. More specifically, it relates to an SMR5 knock out or knock down to improve the oxidative stress tolerance in plants.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/EP2014/074758, filed Nov. 17, 2014, designating the United States of America and published in English as International Patent Publication WO 2015/074992 A1 on May 28, 2015, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. 13193423.4, filed Nov. 19, 2013. 
     
    
     TECHNICAL FIELD 
       [0002]    This application relates generally to plant biology and more specifically to the use of SMR5, possibly in combination with SMR4 and/or SMR7, to modulate ROS and oxidative stress response in plants. More specifically, it relates to an SMR5 knock out or knock down to improve the oxidative stress tolerance in plants. 
       BACKGROUND 
       [0003]    Being immobile, plants are continuously exposed to changing environmental conditions that can impose biotic and abiotic stresses. One of the consequences observed in plants subjected to altered growth conditions is the disruption of the reactive oxygen species (ROS) homeostasis (Mittler et al., 2004). Under steady-state conditions, ROS are efficiently scavenged by different non-enzymatic and enzymatic antioxidant systems, involving the activity of catalases, peroxidases, and glutathione reductases. However, when stress prevails, the ROS production rate can exceed the scavenging mechanisms, resulting into a cell- or tissue-specific rise in ROS. These oxygen derivatives possess a strong oxidizing potential that can damage a wide diversity of biological molecules, including the electron-rich bases of DNA, which results into single- and double-stranded breaks (Amor et al., 1998; Dizdaroglu et al., 2002; Roldan-Mona and Ariza, 2009). Hydrogen peroxide (H 2 O 2 ) is a major ROS compound and is able to transverse cellular membranes, migrating into different compartments. This feature grants H 2 O 2  not only the potential to damage a variety of cellular structures, but also to serve as a signaling molecule, allowing the activation of pathways that modulate developmental, metabolic and defense pathways (Mittler et al., 2011). One of the signaling effects of H 2 O 2  is the activation of a cell division arrest by cell cycle checkpoint activation (Tsukagoshi, 2012), however, the molecular mechanisms involved remain unknown. 
         [0004]    Cell cycle checkpoints adjust cellular proliferation to changing growth conditions, arresting it by the inhibition of the main cell cycle controllers: the heterodimeric complexes between the cyclin-dependent kinases (CDK) and the regulatory cyclins (Lee and Nurse, 1987; Norbury and Nurse, 1992). The activators of these checkpoints are the highly conserved ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR) kinases that are recruited in accordance with the type of DNA damage (Zhou and Elledge, 2000; Abraham, 2001; Bartek and Lukas, 2001; Kurz and Lees-Miller, 2004). ATM is activated by double-stranded breaks (DSBs); whereas ATR is activated by single-stranded breaks or stalled replication forks, causing inhibition of DNA replication. In mammals, ATM and ATR activation result in the phosphorylation of the Chk2 and Chk1 kinases, respectively. In mammals, both kinases subsequently phosphorylate p53, a critical transcription factor responsible to conduct DNA damage responses (Chaturvedi et al., 1999; Shieh et al., 2000; Chen and Sanchez, 2004; Rozan and El-Deiry, 2007). p53 seemingly appears to have no plant ortholog, although an analogous role for p53 is suggested for the plant-specific SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) transcription factor that is under direct post-transcriptional control of ATM (Yoshiyama et al., 2009; Yoshiyama et al., 2013). Another distinct feature relates to the inactivation of CDKs in response to DNA stress. CDK activity is in part controlled by its phosphorylation status at the N-terminus, determined by the interplay of the CDC25 phosphatase and the antagonistic WEE1 kinase, acting as the “on” and “off” switches of CDK activity, respectively (Francis, 2011). Whereas in mammals and budding yeast, the activation of the DNA replication checkpoint, leading to a cell cycle arrest, is predominantly achieved by the inactivation of the CDC25 phosphatase, as plant cells respond to replication stress by transcriptional induction of WEE1 (De Schutter et al., 2007). In absence of WEE1,  Arabidopsis thaliana  plants become hypersensitive to replication inhibitory drugs such as hydroxyurea (HU), which causes a depletion of dNTPs because of an inhibition of the ribonucleotide reductase (RNR) protein. However. WEE1-deficient plants respond similarly to control plants exposed to other types of DNA damage (De Schutter et al., 2007; Dissmeyer et al., 2009). Other, yet to be identified pathways controlling cell cycle progression under DNA stress, operating independently of WEE1, may exist. 
         [0005]    There are several potential candidates to operate in checkpoint activation upon DNA stress mainly belonging to the family of CDK inhibitors (CKIs). CKI proteins are mostly low molecular weight proteins that inhibit cell division by their direct interaction with the CDK and/or cyclin subunit (Sherr and Roberts, 1995; De Clercq and Inzé, 2006). The first identified class of plant CKIs was the ICK/KRP (interactors of CDK/Kip-related protein) protein family comprising seven members in  A. thaliana , all sharing a conserved C-terminal domain being similar to the CDK-binding domain of the animal CIP/KIP proteins (Wang et al., 1998; Wang et al., 2000; De Veylder et al., 2001). The TIC (tissue-specific inhibitors of CDK) is the most recently suggested class of CKIs (DePaoli et al., 2012) and encompasses SCI1 in tobacco, the only tissue-specific CKI reported so far (DePaoli et al., 2011). SCI1 shares no outstanding sequence similarity with the other classes of CKIs in plants, and has been suggested to connect cell cycle progression and auxin signaling in pistils (DePaoli et al., 2012). The third class of CKIs is the plant-specific SIAMESE/SIAMESE-RELATED (SIM/SMR) gene family. SIM has been identified as a cell cycle inhibitor with a role in trichome development and endocycle control (Churchman et al., 2006). Based on sequence analysis, five additional gene family members have been identified in  A. thaliana , and together with EL2 from rice, been suggested to act as cell cycle inhibitors modulated either by biotic and abiotic stresses (Peres et al., 2007). Plants subjected to treatments inducing DSBs showed a rapid and strong induction of specific family members (Culligan et al., 2006; Adachi et al., 2011). 
       SUMMARY OF THE DISCLOSURE 
       [0006]    Surprisingly, it was found that three SMR genes (SMR4, SRM5 and SMR7) are transcriptionally activated by DNA damage. Even more surprisingly, the SMR5 gene encodes for a novel protein not described earlier. Cell cycle inhibitory activity was demonstrated by overexpression analysis, whereas knockout data illustrated that both SMR5 and SMR7 are essential for DNA cell cycle checkpoint activation in leaves of plants grown in the presence of HU. Remarkably, it was found that SMR induction mainly depends on ATM and SOG1, rather than ATR as would be expected for a drug that triggers replication fork defects. Correspondingly, it was demonstrated that the HU-dependent activation of SMR genes is triggered by ROS rather than replication problems, linking SMR genes with cell cycle checkpoint activation upon the occurrence of DNA damage-inducing oxidative stress. 
         [0007]    A first aspect of the disclosure is the use of SMR5, or a homologue, orthologue or paralogue thereof, to modulate ROS signaling and/or oxidative stress response in plants. In a preferred embodiment, this use is combined with the use of SMR4 and/or SMR7. The “use of an SMR,” as used herein, comprises the use of the gene, and/or the use of the protein encoded by the gene. Preferably, the use of SMR5 is the use of a gene encoding a protein selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6 of the incorporated herein Sequence Listing. In one preferred embodiment, the use of SMR5 is the use of a gene encoding a protein preferably consisting of SEQ ID NO:2. In another preferred embodiment, the use of SMR5 is the use of a gene encoding a protein preferably consisting a of a sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:6. “Homologues” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. Orthologues and paralogues encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogues are genes within the same species that have originated through duplication of an ancestral gene; orthologues are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene. 
         [0008]    Preferably, the use is a down-regulation of the expression of the protein, and/or the inactivation of the protein. Preferably, the down-regulation is used to improve oxidative stress tolerance in plants. “Improve” as used herein, means that the plants wherein the SMR is down-regulated have a significantly better oxidative stress resistance than the plants with the same genetic background, except for the modifications needed for the down-regulation, grown under the same conditions. Methods for down-regulation are known to the person skilled in the art, and include, but are not limited to, mutations, insertions or deletions in the gene and/or its promoter, the use of anti-sense RNA or RNAi and gene silencing methods. Methods to induce site-specific mutations in plants are known to the person skilled in the art and include Zinc-finger nucleases, transcription activator-like nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas-based RNA guided DNA endonucleases (Gaj et al., 2013). Inactivation of the protein can be obtained, as a non-limiting example, by the use of antigen-binding proteins directed against the protein, or by protein aggregation, as described in WO 2012/123419. The down-regulation of SMR5 can be measured by measuring the activity of its substrate (Cyclin-dependent kinase A, CDKA) as described in De Veylder et al. (1997); a higher CDKA activity points to a down-regulation of SMR5. 
         [0009]    A “plant” as used herein may be any plant. Plants include gymnosperms and angiosperms, monocotyledons and dicotyledons, trees, fruit trees, field and vegetable crops and ornamental species. Preferably, the plant is a crop plant including, but not limited to, soybean, corn, wheat, barley and rice. 
         [0010]    Another aspect of the disclosure is a genetically modified plant comprising an inactivated SMR5 gene and/or protein. “Inactivated,” as used herein, means that the activity of the inactivated form is significantly lower than that of the active form. “Significantly,” as used herein, means that the activity of the mutant gene or protein is at least 20% lower, preferably at least 50% lower, more preferably at least 75% lower, most preferably at least 90% lower than the wild-type gene or protein. Preferably, the activity of the gene is measured as the amount of messenger RNA. Preferably, the activity of the protein is measured as inhibition of cell division. In one preferred embodiment, the active form of the gene is encoding a protein preferably consisting of SEQ ID NO:2. In another preferred embodiment, the use of SMR5 is the use of a gene encoding a protein preferably consisting of a sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:6. In a preferred embodiment, the plant is a maize plant in which ZmSMRg and/or ZmSMRh are inactivated, preferably as a CRISPR/Cas knock out. 
         [0011]    In one preferred embodiment, the gene encoding the SMR5p is disrupted. In another preferred embodiment, the gene encoding the SMR5p is silenced. In still another embodiment, the SMR5p itself is inactivated by protein aggregation. 
         [0012]    Preferably, the genetically modified plant further comprises an inactivated SMR4 gene and/or protein, and/or an inactivated SMR7 gene and/or protein. 
         [0013]    Still another aspect of the disclosure is a method to increase oxidative stress resistance in a plant comprising the down-regulation of SMR5p expression and/or activity. Preferably, the down-regulation is combined with the down-regulation of SMR4p expression and/or activity, and/or down-regulation of SMR7p expression and/or activity. 
         [0014]    In one preferred embodiment, the method comprises a step wherein the plant is transformed with an RNAi construct against one or more of the SMR genes. In one preferred embodiment, the RNAi construct is placed under control of a constitutive promoter. In another preferred embodiment, the RNAi construct is placed under control of an oxidative stress-inducible promoter. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0015]    The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. 
           [0016]      FIG. 1 : DNA stress meta-analysis. Venn diagram showing the overlap between transcripts induced by hydroxyurea (HU), bleomycin (Bm), and γ-radiation (γ-rays). In total, 61 genes were positively regulated in at least two DNA stress experiments, and 22 genes accumulated in all DNA stress experiments. 
           [0017]      FIGS. 2A and 2B : Hierarchical average linkage clustering of SIM/SMR genes induced in response to different abiotic ( FIG. 2A ) and biotic stresses ( FIG. 2B ). Data comprise the SIM/SMR represented in publicly available AFFYMETRIX® ATHI microarrays obtained with the GENEVESTIGATOR® toolbox. Blue and yellow indicate down- and up-regulation, respectively, whereas black indicates no change in expression. 
           [0018]      FIG. 3 : SIM/SMR induction in response to HU. One-week-old transgenic  Arabidopsis  seedlings were transferred to control (−HU) medium or medium supplemented with 1 mM HU (+HU). GUS assays were performed 24 hours after transfer. 
           [0019]      FIG. 4 : SIM/SMR induction in response to Bleomycine. One-week-old transgenic  Arabidopsis  seedlings were transferred to control (−Bm) medium or medium supplemented with 0.3 μg/mL bleomycin (+Bm). GUS assays were performed after 24 hours after transfer. 
           [0020]      FIG. 5 : Transcriptional induction of SIM/SMR genes upon HU and bleomycin treatment. One-week-old wild-type  Arabidopsis  seedlings were transferred to control medium (blue), or medium supplemented with 1 mM hydroxyurea (red) or 0.3 g/mL bleomycin (green). Root tips were harvested after 24 hours for RT-PCR analysis. Expression levels in control condition were arbitrarily set to one. Data represent mean±SE (n=3). 
           [0021]      FIG. 6 : Transcriptional induction of SIM/SMR genes upon γ-irradiation. (Panels A-F) PSMR4:GUS (Panels A and D), PSMR5:GUS (Panels B and E) and PSMR7:GUS (Panels C and D) either control-treated (Panels A-C) or irradiated with 20 Gy of γ-rays (Panels D-F). GUS assays were performed 1.5 hours after irradiation. 
           [0022]      FIG. 7 : Ectopic SMR4, SMR5 and SMR7 expression inhibits cell division. Panels A-D, Four-week-old rosettes of control (Panel A), SMR4 OE  (Panel B), SMR5 OE  (Panel C), and SMR7 OE  (Panel D) plants. Panels E-H, Leaf abaxial epidermal cell images of in vitro-grown 3-week-old control (Panel E), SMR4 OE  (Panel F), SMR5 OE  (Panel G), and SMR7 OE  (Panel H) plants. Panels I-L, Ploidy level distribution of the first leaves of 3-week-old in vitro-grown control (Panel I), SMR4 OE  (Panel J), SMR5 OE  (Panel K), and SMR7 OE  (Panel L) plants. 
           [0023]      FIGS. 8A and 8B : Graphical representation of the SMR5 and SMR7 T-DNA insertion.  FIG. 8A , Intron-exon organization of the  Arabidopsis  SMR5 and SMR7 genes. Black and white boxes represent coding and non-coding regions, respectively, while lines represent introns. The white triangles indicate the T-DNA insertion sites.  FIG. 8B , qRT-PCR analysis on wild-type, SMR5 KO , SMR7 KO , and SMR5 KO  SMR7 KO  seedlings using primers specific to either SMR5 or SMR7. Expression levels in wild-type were arbitrarily set to one. Data represent mean±SE (n=3). 
           [0024]      FIGS. 9A and 9B : SMR5 and SMR7 are required for an HU-dependent cell cycle checkpoint. Leaf size ( FIG. 9A ) and abaxial epidermal cell number ( FIG. 9B ) of the first leaves of 3-week-old plants grown on control medium (circles) or medium supplemented with 1 mM HU (squares). Data represent mean with 95% confidence interval (n=10). 
           [0025]      FIGS. 10A and 10B : SMR5 and SMR7 expression is ATM- and SOG1-dependent. PSMR5:GUS ( FIG. 10A ) and PSMR7:GUS ( FIG. 10B ) reporter constructs introgressed into atr-2, atm-1 and sog-1 mutant backgrounds were control-treated (Ctrl), or treated with HU or bleomycin (Bm) for 24 hours. 
           [0026]      FIGS. 11A-11C : HU triggers oxidative stress.  FIG. 11A , H 2 O 2  scavenging of control, HU- and 3-AT (positive control) treated plants. Error bars show SEM (n=3-4).  FIG. 11B , Maximum quantum efficiency of PSII (F′v/F′m) of seedlings grown under low (LL) and high light (HL), in absence (−HU) and presence (+HU) of HU.  FIG. 11C , Light microscope pictures of plants shown in  FIG. 11B . 
           [0027]      FIGS. 12A-12D : SMR5 and SMR7 are induced by oxidative stress-inducing stimuli. Relative SMR5 ( FIG. 12A ) and SMR7 ( FIG. 12B ) expression levels in wild-type (Col-0), apx1, cat2 and apx cat2 mutant plants. Expression levels in wild-type were arbitrarily set to one. Data represent mean±SE (n=3).  FIG. 12C , One-week-old PSMR5:GUS and PSMR7:GUS seedlings grown under low-versus high-light conditions.  FIG. 12D , Abaxial epidermal cell number of the first leaves of 3-week-old plants transferred at the age of 8 days for 48 hours to control (circles) or high light (squares) conditions. Data represent mean with 95% confidence interval (n&gt;8). 
           [0028]      FIG. 13 : Cluster analysis of the maize SMR family with the  Arabidopsis  SMR5 
       
    
    
     DETAILED DESCRIPTION 
     Examples 
     Materials and Methods to the Examples 
     Plant Materials and Growth Conditions 
       [0029]    The smr5 (SALK_100918) and smr7 (SALK_128496) alleles were acquired from the  Arabidopsis  Biological Research Center. Homozygous insertion alleles were checked by genotyping PCR using the primers listed in Table 3. The atm-1, atr-2 and sog1-1 mutants have been described previously (Garcia et al., 2003; Preuss and Britt, 2003; Culligan et al., 2004; Yoshiyama et al., 2009). Unless stated otherwise, plants of  Arabidopsis thaliana  (L.) Heyhn (ecotype Columbia), were grown under long-day conditions (16 hours of light, 8 hours of darkness) at 22° C. on half-strength Murashige and Skoog (MS) germination medium (Murashige and Skoog, 1962).  Arabidopsis  plants were treated with HU as described by Cools et al. (2011). For bleomycin treatments, five-day-old seedlings were transferred into liquid MS medium supplemented with 0.3 μg/mL bleomycin. For γ-irradiation treatments, five-day-old in vitro-grown plantlets were irradiated with γ-rays at a dose of 20 Gy. For light treatments, one-week-old seedlings were transferred to continuous high-light conditions (growth rooms kept at 22° C. with 24-hour day/0-hour night cycles and a light intensity of 300-400 μmol m −2  s −1 ) for 2 days, and subsequently retransferred to low-light conditions. The first leaf pair was harvested and incubated in 100% ethanol for epidermis cell drawing as described by De Veylder et al. (2001). 
       DNA and RNA Manipulation 
       [0030]    Genomic DNA was extracted from  Arabidopsis  leaves with the DNEASY® Plant Kit (Qiagen) and RNA was extracted from  Arabidopsis  tissues with the RNEASY® Mini Kit (Qiagen). After DNase treatment with the RQ1 RNase-Free DNase (Promega), cDNA was synthesized with the iScript cDNA Synthesis Kit (Bio-Rad). A quantitative RT-PCR was performed with the SYBR® Green kit (ROCHE) with 100 nM primers and 0.125 μL of RT reaction product in a total of 5μL per reaction. Reactions were run and analyzed on the LIGHTCYCLER® 480 (Roche) according to the manufacturer&#39;s instructions with the use of the following reference genes for normalization: ACTIN2 (At3g46520), EMB2386 (At1g02780), PACI (At3g22110) and RPS26C (At3g56340). Primers used for the RT-PCR are given in Table 5. 
         [0031]    SIM/SMR promoter sequences were amplified from genomic DNA by PCR using the primers described in Table 5. The product fragments were created with the Pfu DNA Polymerase Kit (Promega, Catalog #M7745), and were cloned into a pDONR P4-Plr entry vector by BP recombination cloning and subsequently transferred into the pMK7S*NFml4GW,0 destination vector by LR cloning, resulting in a transcriptional fusion between the promoter of the SMR genes and the nlsGFP-GUS fusion gene (Karimi et al., 2007). For the overexpression constructs, the SMR coding regions were amplified using primers described in Table 5, and cloned into the pDONR221 vector by BP recombination cloning and subsequently transferred into the pK2GW7 destination vector (Kamimi et al., 2002) by LR cloning. All constructs were transferred into the  Agrobacterium tumefaciens  C58C1RifR strain harboring the pMP90 plasmid. The obtained  Agrobacterium  strains were used to generate stably transformed  Arabidopsis  lines with the floral dip transformation method (Clough and Bent, 1998). Transgenic plants were obtained on kanamycin-containing medium and later transferred to soil for optimal seed production. All cloning primers are listed in Table 5. 
       GUS Assays 
       [0032]    Complete seedlings or tissue cuttings were stained in multiwell plates (Falcon 3043; Becton Dickinson). GUS assays were performed as described by Beeckman and Engler (1994). Samples mounted in lactic acid were observed and photographed with a stereomicroscope (Olympus BX51 microscope) or with a differential interference contrast (DIC) microscope (Leica). 
       Microscopy 
       [0033]    For leaf measurements, first leaves were harvested at 21 days after sowing on control medium, medium supplemented with 1 mM hydroxyurea or 0.3 μg/mL bleomycin. Leaves were cleared overnight in ethanol, stored in lactic acid for microscopy, and observed with a microscopy fitted with DIC optics (Leica). The total (blade) area was determined from images digitized directly with a digital camera (Olympus BX51 microscope) mounted on a binocular (Stemi SV 11; Zeiss). From scanned drawing-tube images of the outlines of at least 30 cells of the abaxial epidermis located between 25% to 75% of the distance between the tip and the base of the leaf, halfway between the midrib and the leaf margin, the following parameters were determined: total area of all cells in the drawing and total numbers of pavement and guard cells, from which the average cell area was calculated. The total number of cells per leaf was estimated by dividing the leaf area by the average cell area. For confocal microscopy, root meristems were analyzed 2 days after transfer using a Zeiss LSM 510 Laser Scanning Microscope and the LSM Browser version 4.2 software (Zeiss). Plant material was incubated for 2 minutes in a 10 μm PI solution to stain the cell walls and was visualized with a HeNe laser through excitation at 543 nm. GFP fluorescence was detected with the 488-nm line of an Argon laser. GFP and PI were detected simultaneously by combining the settings indicated above in the sequential scanning facility of the microscope. Acquired images were quantitatively analyzed with the ImageJ v1.45s software (on the World Wide Web at rsbweb.nih.gov/ij/) and Cell-o-Tape plug-ins (French et al., 2012). Chlorophyll a fluorescence parameters were measured using the IMAGING PAM M-Series Chlorofyll Fluorescence (Walz) and associated software. 
       Flow Cytometry Analysis 
       [0034]    For flow cytometric analysis, root tip tissues were chopped with a razor blade in 300 μL of 45 mM MgCl 2 , 30 mM sodium citrate, 20 mM MOPS, pH 7 (Galbraith et al., 1991). One microliter of 4,6-diamidino-2-phenylindole (DAPI) from a stock of 1 mg/mL was added to the filtered supernatant. Leaf material was chopped in 200 μL of Cystain UV Precise P Nuclei extraction buffer (Partec), supplemented with 800 μL of staining buffer. The mix was filtered through a 50-μm green filter and read by the C Y F LOW ® MB flow cytometer (Partec). The nuclei were analyzed with the CYFLOGIC® software. 
       Catalase Assay 
       [0035]    Plants were germinated on either control medium, medium with 1 mM HU or 6 μM 3-AT. Leaf tissue of 10 plants was ground in 200 μL extraction buffer (60 mM Tris (pH 6.9), 1 mM phenylmethylsulfonylfluoride, 10 mM DTT) on ice. The homogenate was centrifuged at 13,000 g for 15 minutes at 4° C. A total of 45 μg protein extract was mixed with potassium phosphate buffer (50 mM, pH 7.0) (Vandenabeele et al., 2004). After addition of 11.4 μL H 2 O 2  (7.5%), the absorbance of the sample at 240 nm after 0 and 60 seconds was measured to determine catalase activity by H 2 O 2  breakdown (Beers and Sizer, 1952; Vandenabeele et al., 2004). 
       Microarray Analysis 
       [0036]    Seeds were plated on sterilized membranes and grown under a 16-hour light/8-hour dark regime at 21° C. After 2 days of germination and 5 days of growth, the membrane was transferred to MS medium containing 0.3 μg/mL bleomycin for 24 hours. Triplicate batches of root meristem material seedlings were harvested for total RNA preparation using the RNEASY® plant mini kit (Qiagen). Each of the different root tip RNA extracts were hybridized to 12 AFFYMETRIX®  Arabidopsis  Gene 1.0 ST Arrays according to manufacturer&#39;s instructions at the Nucleomics Core Facility (Leuven, Belgium; World Wide Web at nucleomics.be). Raw data were processed with the RMA algorithm (Irizarry et al., 2003) using the AFFYMETRIX® Power Tools and subsequently subjected to a Significance Analysis of Microarray (SAM) analysis with “MultiExperiment Viewer 4” (MeV4) of The Institute for Genome Research (TIGR) (Tusher et al., 2001). The imputation engine was set as 10-nearest neighbor imputer and the number of permutations was 100. Expression values were obtained by log 2-transforming the average value of the normalized signal intensities of the triplicate samples. Fold changes were obtained using the expression values of the treatment relative to the control samples. Genes with Q-values&lt;0.1 and fold change&gt;1.5 or &lt;0.666 were retained for further analysis. 
       Microarray Meta-Analysis 
       [0037]    Transcripts induced by bleomycin (Q-value&lt;0.1 and fold change&gt;1.5) were compared with different published DNA stress-related data sets. For γ-irradiation, an intersect of the genes with a significant induction (P-value&lt;0.05, Q-value&lt;0.1, and fold change&gt;1.5) in 5-day-old wild-type seedlings 1.5 hours post-irradiation (100 Gy) was made of two independent experiments (Culligan et al., 2006; Yoshiyama et al., 2009). For replication stress, genes showing a significant induction (P-value (Time)&lt;0.05, Q-value (Time)&lt;0.1 and fold change&gt;1.5) in 5-day-old wild-type root tips after 24 hours of 2-mM hydroxyurea treatment were selected (Cools et al., 2011). Meta-analysis of the SMR genes during various stress conditions and treatments were obtained using GENEVESTIGATOR® (Hruz et al., 2008). Using the “Response Viewer” tool, the expression profiles of genes following different stimuli were analyzed. Only biotic and abiotic stress treatments with a more than 2-fold change in the transcription level (P-value&lt;0.01) for at least one of the SMR genes were taken into account. Fold-change values were hierarchically clustered for genes and experiments by average linkage in MeV from TIGR. 
       Accession Numbers 
       [0038]    Microarray results have been submitted to MiamExpress (on the World Wide Web at ebi.ac.uk/miamexpress), with accession E-MEXP-3977. Sequence data from this article can be found in the  Arabidopsis  Genome Initiative or GenBank/EMBL databases under the following accession numbers: SMR4 (At5g02220); SMR5 (At1g07500); SMR7 (At3g27630); ATM (At3g48490); ATR (At5g40820); and SOG1 (At1g25580). 
       Example 1 
     Meta-Analysis of DNA Stress Datasets Identifies DNA Damage-Induced SMR Genes 
       [0039]    When DNA damage occurs, two global cellular responses are essential for cell survival: activation of the DNA repair machinery and delay or arrest of cell cycle progression. In recent years, gene expression inventories have been collected that focus on the transcriptional changes in response to different types of DNA stress (Culligan et al., 2006; Ricaud et al., 2007; Yoshiyama et al., 2009; Cools et al., 2010). To identify novel key signaling components that contribute to cell cycle checkpoint activation, bleomycin-induced genes were compared to those induced by HU treatment (Cools et al., 2010) and γ-radiation (Culligan et al., 2006; Yoshiyama et al., 2009). Twenty-two genes were up-regulated in all DNA stress experiments and can be considered as transcriptional hallmarks of the DNA damage response (DDR), regardless of the type of DNA stress ( FIG. 1 ; Table 1). Within this selection, genes known to be involved in DNA stress and DNA repair are predominantly present, including PARP2, BRCA1 and RAD51. In addition, one member of the SIM/SMR gene family was recognized, being SMR5 (At1g07500). When expanding the selection by considering genes induced in at least two of the three DNA stress experiments, a total of 61 genes were identified (Table 2). Besides DDR-related genes, this expanded dataset included an additional SMR family member (SMR4; At5g02220), being expressed upon treatment with HU or γ-radiation. 
       Example 2 
     The SMR Gene Family Comprises 14 Family Members that Respond to Different Stresses 
       [0040]    Previously, the existence of one SIM and five SMR genes (SMR1-SMR5) in the  A. thaliana  genome (Peres et al., 2007) was reported, whereas protein purification of CDK/cyclin complexes resulted in the identification of two additional family members (SMR6 and SMR8) (Van Leene et al., 2010). With the availability of new sequenced plant genomes, the  Arabidopsis  genome was re-examined using iterative BLAST searches for the presence of additional SMR genes, resulting in the identification of six non-annotated family members, nominated SMR7 to SMR13 (Table 3). With the GENEVESTIGATOR® toolbox (Hruz et al., 2008), the expression pattern of the twelve SIM/SMR genes represented on the AFFYMETRIX® ATHI microarray platform was analyzed in response to different biotic and abiotic stress treatments. Distinct family members were induced under various stress conditions, albeit with different specificity ( FIGS. 2A and 2B ). Every SMR gene appeared to be transcriptionally active under at least a number of stress conditions, with SMR5 responding to the most diverse types of abiotic stresses. In response to DNA stress (genotoxic stress and UV-B treatment), two SMR genes responded strongly, being SMR4 and SMR5, corresponding with their presence among the DNA stress genes identified by the microarray meta-analysis. 
         [0041]    To confirm involvement of SIM/SMR genes in the genotoxic stress response, transcriptional reporter lines containing the putative upstream promoter sequences were constructed for all. After selection of representative reporter lines, one-week-old seedlings were transferred to control medium, or medium supplemented with HU (resulting into stalled replication forks) or bleomycin (causing DSBs). Focusing on the root tips revealed distinct expression patterns ( FIGS. 3 and 4 ), with some family members being restricted to the root elongation zone (including SIM and SMR1), while others were confined to vascular tissue (e.g., SMR2 and SMR8), or columella cells (e.g., SMR5). When plants were exposed to HU, three SMR genes showed strong transcriptional induction in the root meristem, being SMR4, SMR5 and SMR7, with the latter two displaying the strongest response ( FIG. 3 ). In the presence of bleomycin, an additional weak cell-specific induction of SMR6 was observed ( FIG. 4 ). Transcriptional induction of SMR4, SMR5 and SMR7 by HU and bleomycin was confirmed by qRT-PCR experiments ( FIG. 5 ). These data fit the above-described microarray analysis, with the lack of SMR7 (At3g27630) being explained by its absence on the ATHI microarray of the HU and γ-irradiation experiments, although being induced 5.68-fold in the bleomycin experiment performed using the Aragene array. Next to HU and bleomycin, transcriptional activation of SMR4, SMR5 and SMR7 was confirmed by γ-irradiation ( FIG. 6 ). 
       Example 3 
     DNA Stress-Induced SMR Genes Encode Potent Cell Cycle Inhibitors 
       [0042]    Previously, SIM had been proven to encode a potent cell cycle inhibitor, since its ectopic expression results in dwarf plants holding less cells compared to control plants (Churchman et al., 2006). To test whether the DNA stress-induced SMR genes encode proteins with cell division inhibitory activity, SMR4-, SMR5- and SMR7-overexpressing (SMR4 OE , SMR5 OE  and SMR7 OE ) plants were generated. For each gene, multiple lines with strong transcript levels were isolated, all showing a reduction in rosette size compared to wild-type plants ( FIG. 7 , Panels A to D). This decrease in leaf size correlated with an increase in cell size ( FIG. 7 , Panels E to H), indicative of a strong inhibition of cell division. Similar to SIM (Churchman et al., 2006), ectopic expression did not only inhibit cell division but also triggered an increase in the DNA content by stimulation of endoreplication ( FIG. 7 , Panels I to L; Table 4), likely representing a premature onset of cell differentiation. Together with the previously described biochemical interaction between SMR4 and SMR5, and CDKA;1 and D-type cyclins (Van Leene et al., 2010), it can be concluded that the DNA stress-induced SMR genes encode potent cell cycle inhibitors. 
       Example 4 
     SMR5 and SMR7 Control an HU-Dependent Checkpoint in Leaves 
       [0043]    To address the role of the different SMR genes in DNA stress checkpoint control, the growth response to HU treatment of plants being knocked out for SMR5 or SMR7 ( FIGS. 8A and 8B ) was compared to that of control plants (Col-0). No significant difference in leaf size was observed for plants grown under standard conditions. In contrast, when comparing plants grown for 3 weeks in the presence of HU, the size of the SMR5 KO  and SMR7 KO  leaves was significantly bigger than that of the control plants ( FIG. 9A ). This difference was attributed to a difference in cell number. Control plants responded to the HU treatment with a 47% reduction in epidermal cell number, reflecting an activation of a stringent cell cycle checkpoint. In contrast, in SMR5 KO  and SMR7 KO  plants, this reduction was restricted to 29% and 30%, respectively ( FIG. 9B ). Within the SMR5 KO  SMR7 KO  double mutant, the reduction in leaf size and cell number was even less ( FIGS. 9A and 9B ), suggesting that both inhibitors contribute to the cell cycle arrest observed in the control plants by checkpoint activation upon HU stress. Unfortunately, a similar role of SMR4 could not be tested due to the lack of an available knockout. 
       Example 5 
     SMR5 and SMR7 Expression is Triggered by Oxidative Stress 
       [0044]    Because of the observed role of the SMR5 and SMR7 genes in DNA stress checkpoint control, the dependence of their expression on the ATM and ATR signaling kinases and the SOG1 transcription factor was analyzed by introducing the SMR5 and SMR7 GUS reporter lines into the atr-2, atm-1 and sog1-1 mutant backgrounds. Both genes were induced in the proliferating leaf upon HU and bleomycin treatment ( FIGS. 10A and 10B ). Moreover, as would be expected for a DSB-inducing agent, the transcriptional activation of SMR5 and SMR7 by bleomycin depended on ATM and SOG1. Surprisingly, the same pattern was observed for HU, whereas one would expect that SMR5/SMR7 induction after arrest of the replication fork would rely on ATR-dependent signaling. These data indicate that the HU-dependent activation of the SMR5 and SMR7 genes might be caused by a genotoxic effect of HU being unrelated to replication stress induced by the depletion of dNTPs. A recent study demonstrated that HU directly inhibits catalase-mediated H 2 O 2  decomposition (Juul et al., 2010). Analogously, in combination with H 2 O 2 , HU has been demonstrated to act as a suicide inhibitor of ascorbate peroxidase (Chen and Asada, 1990). Combined, both mechanisms are likely responsible for an increase in the cellular H 2 O 2  concentration, which might trigger DNA damage and consequently transcriptional induction of the SMR5 and SMR7 genes. Indeed, extracts of control plants treated with HU displayed a reduced H 2 O 2  decomposition rate ( FIG. 11A ). As catalase and ascorbate peroxidase activity are essential for the scavenging of H 2 O 2  that is generated upon high-light exposure, the effects of HU treatment on photosystem II (PSII) efficiency in one-week-old seedlings was subsequently tested after transfer from low- to high-light conditions. As illustrated in  FIG. 11B , transfer for 48 hours to high light resulted in a decrease of maximum quantum efficiency of PSII (F′v/F′m). In the presence of HU, the F′v/F′m decrease was even more pronounced, which again corroborates the idea that HU might interfere with H 2 O 2  scavenging. Macroscopically, plants grown in the presence of HU accumulated anthocyanins in the young leaf tissue within 48 hours after transfer, whereas plants grown on control medium showed no effect of the transfer to high light ( FIG. 11C ). 
         [0045]    To examine whether an increase in H 2 O 2  might trigger expression of SMR genes, SMR5 and SMR7 expression levels were analyzed in plants that are knockout for CAT2 and/or APX1, encoding two enzymes important for the scavenging of H 2 O 2 . SMR5 expression levels were clearly induced in the apx1 cat2 double mutant, whereas SMR7 transcriptional activation was observed in the apx1 knockout and apx1 cat2 double mutant ( FIG. 12A ). Analogously, plants grown for two days under high light conditions displayed PSMR5:GUS and SMR7:GUS induction in proliferating leaves ( FIG. 12B ). To examine whether this transcriptional induction contributed to a high light-induced cell cycle checkpoint, the epidermal cell numbers were measured in mature first leaves of control (Col-0), SMR5 KO  and SMR7 KO  plants that were transferred for two days to high light condition at the moment that their leaves were proliferating. This high light treatment resulted into a 34% and 38% reduction in cell number in control and SMR7 KO  plants, respectively ( FIG. 12C ). In contrast, SMR5 K0  plants displayed only a 13% reduction in cell number, illustrating that SMR5 is essential to activate a high light-dependent cell cycle checkpoint. 
       Example 6 
     Identification of Maize SMR5 Orthologues 
       [0046]    Sequences of the  Arabidopsis  and maize SMR proteins were aligned and subsequently clustered. The maize proteins ZmSMRg and ZmSMRh were identified as the closest orthologues of  Arabidopsis  SMR5. The coding sequence is given in SEQ ID NO:3 (ZmSMRg) and SEQ ID NO:5 (ZmSMRh). The results are given in  FIG. 13 . 
         [0047]    The transcriptional induction of the maize SMR genes after HU treatment was measured using qRT-PCR analysis, similar as described for  Arabidopsis , and both genes show a strong up-regulation upon HU treatment, both in root tips and in leaves. 
         [0048]    Detailed expression analysis of both the ZmSMRg gene and the ZmSMRh gene is carried out using promoter-GUS fusions, transformed into maize. These transformed plants are tested under a variety of stresses including, but not limited to, drought, high light, cold, heat, hydroxyurea and bleomycin treatment. 
       Example 7 
     Knock Out Mutants in Maize 
       [0049]    The ZmSMRg gene and the ZmSMRh gene are knocked out using the CRISPR-Cas technology, generating single and double knock out mutants. These knock out mutants are submitted to oxidative stress as described for  Arabidopsis , and the mutants show a significant protection against oxidative stress, when compared to the wild-type grown under the same conditions. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Overview of the transcriptionally induced core DNA damage 
               
               
                 genes 
               
             
          
           
               
                   
                   
                 HU 
                 γ-rays -  
                 γ-rays -  
                   
               
               
                 AGI locus 
                 Annotation 
                 24 h/0 h a   
                 1 b   
                 2 c   
                 Bleomycin 
               
               
                   
               
             
          
           
               
                 AT4G21070 
                 Breast cancer susceptibility1 
                 10.375 
                 581.570 
                 57.803 
                 2.386 
               
               
                 AT5G60250 
                 Zinc finger (C3HC4-type RING finger) 
                 8.907 
                 34.918 
                 40.000 
                 2.352 
               
               
                   
                 family protein 
               
               
                 AT1G07500 
                 Siamese-related 5 
                 7.863 
                 38.160 
                 35.842 
                 1.595 
               
               
                 AT4G02390 
                 Poly(ADP-ribose) polymerase 
                 7.701 
                 131.865 
                 59.172 
                 2.663 
               
               
                 AT3G07800 
                 Thymidine kinase 
                 7.160 
                 46.179 
                 20.492 
                 2.759 
               
               
                 AT5G03780 
                 TRF-like 10 
                 7.111 
                 108.316 
                 23.474 
                 1.600 
               
               
                 AT5G64060 
                 NAC domain containing protein 103 
                 5.579 
                 28.086 
                 13.755 
                 2.153 
               
               
                 AT2G18600 
                 Ubiquitin-conjugating enzyme family 
                 5.521 
                 21.462 
                 11.481 
                 1.972 
               
               
                   
                 protein 
               
               
                 AT4G22960 
                 Unknown function (DUF544) 
                 5.315 
                 36.380 
                 14.451 
                 2.282 
               
               
                 AT5G48720 
                 X-ray induced transcript 1 
                 5.296 
                 285.166 
                 65.789 
                 2.228 
               
               
                 AT5G24280 
                 Gamma-irradiation and mitomycin c 
                 4.823 
                 108.578 
                 42.918 
                 2.584 
               
               
                   
                 induced 1 
               
               
                 AT5G20850 
                 RAS associated with diabetes protein 
                 4.643 
                 186.456 
                 31.250 
                 1.765 
               
               
                   
                 51 
               
               
                 AT3G27060 
                 Ferritin/ribonucleotide reductase-like 
                 4.595 
                 37.351 
                 8.741 
                 1.970 
               
               
                   
                 family protein 
               
               
                 AT2G46610 
                 RNA-binding (RRM/RBD/RNP motifs) 
                 3.593 
                 19.913 
                 7.331 
                 1.546 
               
               
                   
                 family protein 
               
               
                 AT5G40840 
                 Rad21/Rec8-like family protein 
                 3.375 
                 113.919 
                 27.473 
                 1.692 
               
               
                 AT1G13330 
                 Hop2 homolog 
                 2.949 
                 17.349 
                 13.495 
                 1.580 
               
               
                 AT5G66130 
                 RADIATION SENSITIVE 17 
                 2.888 
                 30.411 
                 10.384 
                 1.627 
               
               
                 AT1G17460 
                 TRF-like 3 
                 2.378 
                 18.925 
                 10.661 
                 1.681 
               
               
                 AT2G45460 
                 SMAD/FHA domain-containing protein 
                 2.378 
                 45.673 
                 21.053 
                 1.575 
               
               
                 AT5G49480 
                 Ca2+-binding protein 1 
                 1.952 
                 15.106 
                 5.851 
                 1.580 
               
               
                 AT3G25250 
                 AGC (cAMP-dependent, cGMP- 
                 1.853 
                 12.995 
                 17.794 
                 1.517 
               
               
                   
                 dependent and protein kinase C) kinase 
               
               
                   
                 family protein 
               
               
                 AT5G55490 
                 Gamete expressed protein 1 
                 1.670 
                 71.489 
                 34.722 
                 2.407 
               
               
                   
               
               
                   a According to Cools et al., 2011 
               
               
                   b According to Culligan et al., 2006 
               
               
                   c According to Yoshiyama et al., 2009 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Meta-analysis of genes induced in multiple DNA damage experiments. 
               
             
          
           
               
                   
                   
                 q- 
                 p- 
                   
                 q- 
                 p- 
                   
                 q- 
                 p- 
                   
                 q- 
                   
               
               
                   
                   
                 value 
                 value 
                   
                 value 
                 value 
                   
                 value 
                 value 
                   
                 value 
               
               
                   
                   
                 (HU - 
                 (HU - 
                 HU 
                 (γ-rays - 
                 (γ-rays - 
                 γ-rays - 
                 (γ-rays - 
                 (γ-rays - 
                 γ-rays - 
                 Bleo- 
                 Bleo- 
               
               
                 Locus 
                 Description 
                 Time) a   
                 Time) a   
                 24 h/0 h a   
                 1) b   
                 1) b   
                 1 b   
                 2) c   
                 2) c   
                 2 c   
                 mycin 
                 mycin 
               
               
                   
               
             
          
           
               
                 Significantly 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Induced by 
               
               
                 HU, BM and 
               
               
                 gammarays 
               
               
                 AT4G21070 
                 breast cancer 
                 0.018 
                 0.001 
                 10.375 
                 0.000 
                 0.000 
                 581.570 
                 0.000 
                 0.000 
                 57.803 
                 0.000 
                 2.386 
               
               
                   
                 susceptibility1 
               
               
                 AT5G60250 
                 zinc finger 
                 0.000 
                 0.000 
                 8.907 
                 0.001 
                 0.000 
                 34.918 
                 0.000 
                 0.000 
                 40.000 
                 0.000 
                 2.352 
               
               
                   
                 (C3HC4-type 
               
               
                   
                 RING finger) 
               
               
                   
                 family protein 
               
               
                 AT1G07500 
                 unknown 
                 0.000 
                 0.000 
                 7.863 
                 0.003 
                 0.000 
                 38.160 
                 0.000 
                 0.001 
                 35.842 
                 0.000 
                 1.595 
               
               
                   
                 protein; Has 4 
               
               
                   
                 Blast hits to 4 
               
               
                   
                 proteins in 3 
               
               
                   
                 species: 
               
               
                   
                 Archae - 0; 
               
               
                   
                 Bacteria - 0; 
               
               
                   
                 Metazoa - 0; 
               
               
                   
                 Fungi - 0; 
               
               
                   
                 Plants - 4; 
               
               
                   
                 Viruses - 0; 
               
               
                   
                 Other 
               
               
                   
                 Eukaryotes - 0 
               
               
                   
                 (source: 
               
               
                   
                 NCBI BLink). 
               
               
                 AT4G02390 
                 poly(ADP- 
                 0.000 
                 0.000 
                 7.701 
                 0.001 
                 0.000 
                 131.865 
                 0.000 
                 0.000 
                 59.172 
                 0.000 
                 2.663 
               
               
                   
                 ribose) 
               
               
                   
                 polymerase 
               
               
                 AT3G07800 
                 Thymidine 
                 0.033 
                 0.002 
                 7.160 
                 0.000 
                 0.000 
                 46.179 
                 0.000 
                 0.004 
                 20.492 
                 0.000 
                 2.759 
               
               
                   
                 kinase 
               
               
                 AT5G03780 
                 TRF-like 10 
                 0..018 
                 0.001 
                 7.111 
                 0.005 
                 0.000 
                 108.316 
                 0.000 
                 0.003 
                 23.474 
                 0.036 
                 1.600 
               
               
                 AT5G64060 
                 NAC domain 
                 0.014 
                 0.000 
                 5.579 
                 0.004 
                 0.000 
                 28.086 
                 0.000 
                 0.008 
                 13.755 
                 0.002 
                 2.153 
               
               
                   
                 containing 
               
               
                   
                 protein 103 
               
               
                 AT2G18600 
                 Ubiquitin- 
                 0.009 
                 0.000 
                 5.521 
                 0.004 
                 0.000 
                 21.462 
                 0.000 
                 0.014 
                 11.481 
                 0.004 
                 1.972 
               
               
                   
                 conjugating 
               
               
                   
                 enzyme 
               
               
                   
                 family protein 
               
               
                 AT4G22960 
                 Protein of 
                 0.012 
                 0.000 
                 5.315 
                 0.009 
                 0.000 
                 36.380 
                 0.000 
                 0.009 
                 14.451 
                 0.000 
                 2.282 
               
               
                   
                 unknown 
               
               
                   
                 function 
               
               
                   
                 (DUF544) 
               
               
                 AT5G48720 
                 x-ray induced 
                 0.048 
                 0.003 
                 5.296 
                 0.004 
                 0.000 
                 285.166 
                 0.000 
                 0.000 
                 65.789 
                 0.000 
                 2.228 
               
               
                   
                 transcript 1 
               
               
                 AT5G24280 
                 gamma- 
                 0.026 
                 0.001 
                 4.823 
                 0.009 
                 0.000 
                 108.578 
                 0.000 
                 0.000 
                 42.918 
                 0.000 
                 2.584 
               
               
                   
                 irradiation and 
               
               
                   
                 mitomycin c 
               
               
                   
                 induced 1 
               
               
                 AT5G20850 
                 RAS 
                 0.031 
                 0.002 
                 4.643 
                 0.002 
                 0.000 
                 186.456 
                 0.000 
                 0.001 
                 31.250 
                 0.000 
                 1.765 
               
               
                   
                 associated 
               
               
                   
                 with diabetes 
               
               
                   
                 protein 51 
               
               
                 AT3G27060 
                 Ferritin/ribonucleotide 
                 0.012 
                 0.000 
                 4.595 
                 0.001 
                 0.000 
                 37.351 
                 0.000 
                 0.018 
                 8.741 
                 0.000 
                 1.970 
               
               
                   
                 reductase-like 
               
               
                   
                 family protein 
               
               
                 AT2G46610 
                 RNA-binding 
                 0.027 
                 0.001 
                 3.593 
                 0.002 
                 0.000 
                 19.913 
                 0.000 
                 0.021 
                 7.331 
                 0.021 
                 1.546 
               
               
                   
                 (RRM/RBD/RNP 
               
               
                   
                 motifs) 
               
               
                   
                 family protein 
               
               
                 AT5G40840 
                 Rad21/Rec8- 
                 0.052 
                 0.004 
                 3.375 
                 0.005 
                 0.000 
                 113.919 
                 0.000 
                 0.002 
                 27.473 
                 0.002 
                 1.692 
               
               
                   
                 like family 
               
               
                   
                 protein 
               
               
                 AT1G13330 
                 
                   Arabidopsis 
                 
                 0.014 
                 0.000 
                 2.949 
                 0.019 
                 0.000 
                 17.349 
                 0.000 
                 0.009 
                 13.495 
                 0.046 
                 1.580 
               
               
                   
                 Hop2 
               
               
                   
                 homolog 
               
               
                 AT5G66130 
                 Radiation 
                 0.009 
                 0.000 
                 2.888 
                 0.003 
                 0.000 
                 30.411 
                 0.000 
                 0.015 
                 10.384 
                 0.002 
                 1.627 
               
               
                   
                 Sensitive 17 
               
               
                 AT1G17460 
                 TRF-like 3 
                 0.052 
                 0.004 
                 2.378 
                 0.000 
                 0.000 
                 18.925 
                 0.000 
                 0.015 
                 10.661 
                 0.007 
                 1.681 
               
               
                 AT2G45460 
                 SMAD/FHA 
                 0.012 
                 0.000 
                 2.378 
                 0.000 
                 0.000 
                 45.673 
                 0.000 
                 0.004 
                 21.053 
                 0.010 
                 1.575 
               
               
                   
                 domain- 
               
               
                   
                 containing 
               
               
                   
                 protein 
               
               
                 AT5G49480 
                 Ca2+-binding 
                 0.021 
                 0.001 
                 1.952 
                 0.002 
                 0.000 
                 15.106 
                 0.000 
                 0.026 
                 5.851 
                 0.010 
                 1.580 
               
               
                   
                 protein 1 
               
               
                 AT3G25250 
                 AGC (cAMP- 
                 0.014 
                 0.000 
                 1.853 
                 0.003 
                 0.000 
                 12.995 
                 0.000 
                 0.004 
                 17.794 
                 0.035 
                 1.517 
               
               
                   
                 dependent, 
               
               
                   
                 cGMP- 
               
               
                   
                 dependent and 
               
               
                   
                 protein kinase 
               
               
                   
                 C) kinase 
               
               
                   
                 family protein 
               
               
                 AT5G55490 
                 gamete 
                 0.034 
                 0.002 
                 1.670 
                 0.000 
                 0.000 
                 71.489 
                 0.000 
                 0.001 
                 34.722 
                 0.000 
                 2.407 
               
               
                   
                 expressed 
               
               
                   
                 protein 1 
               
               
                 Significantly 
               
               
                 induced by 
               
               
                 HU and 
               
               
                 gamma rays 
               
               
                 AT4G28950 
                 RHO-related 
                 0.021 
                 0.001 
                 9.680 
                 0.000 
                 0.000 
                 36.081 
                 0.000 
                 0.008 
                 13.569 
               
               
                   
                 protein from 
               
               
                   
                 plants 9 
               
               
                 AT3G45730 
                 unknown 
                 0.034 
                 0.002 
                 5.637 
                 0.000 
                 0.000 
                 46.290 
                 0.000 
                 0.009 
                 14.286 
               
               
                   
                 protein; Has 3 
               
               
                   
                 Blast hits to 3 
               
               
                   
                 proteins in 1 
               
               
                   
                 species: 
               
               
                   
                 Archae - 0; 
               
               
                   
                 Bacteria - 0; 
               
               
                   
                 Metazoa - 0; 
               
               
                   
                 Fungi - 0; 
               
               
                   
                 Plants - 3; 
               
               
                   
                 Viruses - 0; 
               
               
                   
                 Other 
               
               
                   
                 Eukaryotes - 0 
               
               
                   
                 (source: 
               
               
                   
                 NCBI BLink). 
               
               
                 AT5G11460 
                 Protein of 
                 0.006 
                 0.000 
                 5.483 
                 0.003 
                 0.000 
                 41.596 
                 0.000 
                 0.005 
                 16.863 
               
               
                   
                 unknown 
               
               
                   
                 function 
               
               
                   
                 (DUF581) 
               
               
                 AT5G02220 
                 unknown 
                 0.023 
                 0.001 
                 4.500 
                 0.001 
                 0.000 
                 45.759 
                 0.000 
                 0.004 
                 20.534 
               
               
                   
                 protein; Has 
               
               
                   
                 30201 Blast 
               
               
                   
                 hits to 17322 
               
               
                   
                 proteins in 
               
               
                   
                 780 species: 
               
               
                   
                 Archae - 12; 
               
               
                   
                 Bacteria - 1396; 
               
               
                   
                 Metazoa - 17338; 
               
               
                   
                 Fungi - 3422; 
               
               
                   
                 Plants - 5037; 
               
               
                   
                 Viruses - 0; 
               
               
                   
                 Other 
               
               
                   
                 Eukaryotes - 2996 
               
               
                   
                 (source: 
               
               
                   
                 NCBI BLink). 
               
               
                 AT2G47680 
                 zinc finger 
                 0.031 
                 0.002 
                 3.422 
                 0.022 
                 0.000 
                 50.849 
                 0.000 
                 0.004 
                 17.513 
               
               
                   
                 (CCCH type) 
               
               
                   
                 helicase 
               
               
                   
                 family protein 
               
               
                 AT4G29170 
                 Mnd1 family 
                 0.060 
                 0.005 
                 2.898 
                 0.000 
                 0.000 
                 40.733 
                 0.000 
                 0.006 
                 16.694 
               
               
                   
                 protein 
               
               
                 AT5G06190 
                 unknown 
                 0.012 
                 0.000 
                 2.878 
                 0.008 
                 0.007 
                 3.757 
                 0.001 
                 0.092 
                 2.690 
               
               
                   
                 protein; BEST 
               
               
                   
                 
                   Arabidopsis 
                 
               
               
                   
                 
                   thaliana 
                 
               
               
                   
                 protein match 
               
               
                   
                 is: unknown 
               
               
                   
                 protein 
               
               
                   
                 (TAIR: AT3G58540.1); 
               
               
                   
                 Has 30201 Blast 
               
               
                   
                 hits to 17322 
               
               
                   
                 proteins in 
               
               
                   
                 780 species: 
               
               
                   
                 Archae - 12; 
               
               
                   
                 Bacteria - 1396; 
               
               
                   
                 Metazoa - 17338; 
               
               
                   
                 Fungi - 3422; 
               
               
                   
                 Plants - 5037; 
               
               
                   
                 Viruses - 0; 
               
               
                   
                 Other 
               
               
                   
                 Eukaryote 
               
               
                 AT5G67460 
                 O-Glycosyl 
                 0.031 
                 0.002 
                 2.799 
                 0.005 
                 0.000 
                 18.032 
                 0.000 
                 0.004 
                 17.271 
               
               
                   
                 hydrolases 
               
               
                   
                 family 17 
               
               
                   
                 protein 
               
               
                 AT4G35740 
                 DEAD/DEAH 
                 0.037 
                 0.002 
                 2.594 
                 0.002 
                 0.000 
                 21.434 
                 0.000 
                 0.021 
                 7.037 
               
               
                   
                 box RNA 
               
               
                   
                 helicase 
               
               
                   
                 family protein 
               
               
                 AT2G21790 
                 ribonucleotide 
                 0.045 
                 0.003 
                 2.514 
                 0.000 
                 0.000 
                 13.702 
                 0.000 
                 0.034 
                 4.948 
               
               
                   
                 reductase 1 
               
               
                   
                 SMAD/FHA 
               
               
                 AT3G02400 
                 domain- 
                 0.052 
                 0.004 
                 2.479 
                 0.025 
                 0.002 
                 9.474 
                 0.000 
                 0.022 
                 6.649 
               
               
                   
                 containing 
               
               
                   
                 protein 
               
               
                 AT2G31320 
                 poly(ADP- 
                 0.020 
                 0.001 
                 2.445 
                 0.001 
                 0.000 
                 39.238 
                 0.000 
                 0.015 
                 9.970 
               
               
                   
                 ribose) 
               
               
                   
                 polymerase 2 
               
               
                 AT3G42860 
                 zinc knuckle 
                 0.039 
                 0.002 
                 2.445 
                 0.001 
                 0.000 
                 30.770 
                 0.000 
                 0.010 
                 13.351 
               
               
                   
                 (CCHC-type) 
               
               
                   
                 family protein 
               
               
                 AT1G09815 
                 polymerase 
                 0.026 
                 0.001 
                 2.354 
                 0.000 
                 0.000 
                 19.771 
                 0.000 
                 0.021 
                 7.310 
               
               
                   
                 delta 4 
               
               
                 AT3G20490 
                 unknown 
                 0.043 
                 0.003 
                 2.313 
                 0.003 
                 0.000 
                 17.593 
                 0.000 
                 0.029 
                 5.291 
               
               
                   
                 protein; Has 
               
               
                   
                 754 Blast hits 
               
               
                   
                 to 165 
               
               
                   
                 proteins in 64 
               
               
                   
                 species: 
               
               
                   
                 Archae - 0; 
               
               
                   
                 Bacteria - 48; 
               
               
                   
                 Metazoa - 26; 
               
               
                   
                 Fungi - 25; 
               
               
                   
                 Plants - 36; 
               
               
                   
                 Viruses - 0; 
               
               
                   
                 Other 
               
               
                   
                 Eukaryotes - 619 
               
               
                   
                 (source: 
               
               
                   
                 NCBI BLink). 
               
               
                 AT4G19130 
                 Replication 
                 0.093 
                 0.010 
                 2.305 
                 0.010 
                 0.000 
                 59.037 
                 0.000 
                 0.010 
                 13.089 
               
               
                   
                 factor-A 
               
               
                   
                 protein 1- 
               
               
                   
                 related 
               
               
                 AT2G30360 
                 SOS3- 
                 0.033 
                 0.002 
                 2.274 
                 0.004 
                 0.000 
                 11.137 
                 0.000 
                 0.017 
                 9.346 
               
               
                   
                 interacting 
               
               
                   
                 protein 4 
               
               
                 AT3G12510 
                 MADS-box 
                 0.006 
                 0.000 
                 2.266 
                 0.001 
                 0.000 
                 17.935 
                 0.000 
                 0.029 
                 5.426 
               
               
                   
                 family protein 
               
               
                 AT1G12020 
                 unknown 
                 0.030 
                 0.001 
                 1.873 
                 0.006 
                 0.000 
                 8.806 
                 0.001 
                 0.080 
                 2.976 
               
               
                   
                 protein; BEST 
               
               
                   
                 
                   Arabidopsis 
                 
               
               
                   
                 
                   thaliana 
                 
               
               
                   
                 protein match 
               
               
                   
                 is: unknown 
               
               
                   
                 protein 
               
               
                   
                 (TA1R: AT1G62422.1); 
               
               
                   
                 Has 89 Blast hits 
               
               
                   
                 to 88 proteins 
               
               
                   
                 in 16 species: 
               
               
                   
                 Archae - 0; 
               
               
                   
                 Bacteria - 0; 
               
               
                   
                 Metazoa - 0; 
               
               
                   
                 Fungi - 0; 
               
               
                   
                 Plants - 87; 
               
               
                   
                 Viruses - 0; 
               
               
                   
                 Other 
               
               
                   
                 Eukaryotes - 2 
               
               
                   
                 (source: NCBI 
               
               
                 AT1G31280 
                 Argonaute 
                 0.014 
                 0.000 
                 1.866 
                 0.002 
                 0.000 
                 24.264 
                 0.000 
                 0.017 
                 9.302 
               
               
                   
                 family protein 
               
               
                 AT1G59660 
                 Nucleoporin 
                 0.033 
                 0.002 
                 1.860 
                 0.014 
                 0.000 
                 15.946 
                 0.000 
                 0.013 
                 11.933 
               
               
                   
                 autopeptidase 
               
               
                 AT3G15240 
                 Serine/threonine- 
                 0.027 
                 0.001 
                 1.790 
                 0.016 
                 0.001 
                 6.471 
                 0.001 
                 0.060 
                 3.552 
               
               
                   
                 protein 
               
               
                   
                 kinase WNK 
               
               
                   
                 (With No 
               
               
                   
                 Lysine)- 
               
               
                   
                 related 
               
               
                 AT1G30600 
                 Subtilase 
                 0.093 
                 0.010 
                 1.711 
                 0.013 
                 0.000 
                 9.920 
                 0.001 
                 0.066 
                 3.299 
               
               
                   
                 family protein 
               
               
                 AT5G67360 
                 Subtilase 
                 0.029 
                 0.001 
                 1.676 
                 0.001 
                 0.000 
                 4.720 
                 0.001 
                 0.082 
                 2.923 
               
               
                   
                 family protein 
               
               
                 AT1G76180 
                 Dehydrin 
                 0.062 
                 0.005 
                 1.659 
                 0.017 
                 0.010 
                 3.048 
                 0.001 
                 0.080 
                 2.975 
               
               
                   
                 family protein 
               
               
                 AT4G11740 
                 Ubiquitin-like 
                 0.084 
                 0.008 
                 1.653 
                 0.000 
                 0.000 
                 7.747 
                 0.001 
                 0.067 
                 3.272 
               
               
                   
                 superfamily 
               
               
                   
                 protein 
               
               
                 AT2G36910 
                 ATP binding 
                 0.012 
                 0.000 
                 1.569 
                 0.000 
                 0.001 
                 3.596 
                 0.001 
                 0.092 
                 2.693 
               
               
                   
                 cassette 
               
               
                   
                 subfamily B1 
               
               
                 AT5G14930 
                 senescence- 
                 0.000 
                 0.000 
                 1.542 
                 0.000 
                 0.000 
                 9.606 
                 0.000 
                 0.018 
                 8.993 
               
               
                   
                 associated 
               
               
                   
                 gene 101 
               
               
                 Significantly 
               
               
                 induced by 
               
               
                 HU and BM 
               
               
                 AT5G66985 
                 unknown 
                 0.088 
                 0.009 
                 3.294 
                   
                   
                   
                   
                   
                   
                 0.007 
                 1.612 
               
               
                   
                 protein; Has 
               
               
                   
                 30201 Blast 
               
               
                   
                 hits to 17322 
               
               
                   
                 proteins in 
               
               
                   
                 780 species: 
               
               
                   
                 Archae - 12; 
               
               
                   
                 Bacteria - 1396; 
               
               
                   
                 Metazoa - 17338; 
               
               
                   
                 Fungi - 3422; 
               
               
                   
                 Plants - 5037; 
               
               
                   
                 Viruses - 0; 
               
               
                   
                 Other 
               
               
                   
                 Eukaryotes - 2996 
               
               
                   
                 (source: 
               
               
                   
                 NCBI BLink). 
               
               
                 AT5G14920 
                 Gibberellin- 
                 0.027 
                 0.001 
                 2.789 
                   
                   
                   
                   
                   
                   
                 0.000 
                 2.122 
               
               
                   
                 regulatcd 
               
               
                   
                 family protein 
               
               
                 AT4G15480 
                 UDP- 
                 0.081 
                 0.008 
                 2.196 
                   
                   
                   
                   
                   
                   
                 0.000 
                 2.394 
               
               
                   
                 Glycosyltransferase 
               
               
                   
                 superfamily 
               
               
                   
                 protein 
               
               
                 AT3G27620 
                 alternative 
                 0.077 
                 0.007 
                 2.056 
                   
                   
                   
                   
                   
                   
                 0.025 
                 1.883 
               
               
                   
                 oxidase 1C 
               
               
                 AT3G27950 
                 GDSL-like 
                 0.045 
                 0.003 
                 1.641 
                   
                   
                   
                   
                   
                   
                 0.000 
                 4.012 
               
               
                   
                 Lipase/Acylhydrolase 
               
               
                   
                 superfamily 
               
               
                   
                 protein 
               
               
                 AT4G04750 
                 Major 
                 0.082 
                 0.008 
                 1.625 
                   
                   
                   
                   
                   
                   
                 0.011 
                 1.689 
               
               
                   
                 facilitator 
               
               
                   
                 superfamily 
               
               
                   
                 protein 
               
               
                 AT5G60100 
                 pseudo- 
                 0.037 
                 0.002 
                 1.619 
                   
                   
                   
                   
                   
                   
                 0.018 
                 1.801 
               
               
                   
                 response 
               
               
                   
                 regulator 3 
               
               
                 AT5G25810 
                 Integrase-type 
                 0.000 
                 0.000 
                 1.558 
                   
                   
                   
                   
                   
                   
                 0.040 
                 1.573 
               
               
                   
                 DNA-binding 
               
               
                   
                 superfamily 
               
               
                   
                 protein 
               
               
                 AT1G49030 
                 PLAC8 family 
                 0.044 
                 0.003 
                 1.553 
                   
                   
                   
                   
                   
                   
                 0.000 
                 2.653 
               
               
                   
                 protein 
               
               
                 Significantly 
               
               
                 induced by 
               
               
                 BM and 
               
               
                 gamma rays 
               
               
                 AT4G05370 
                 BCS1 AAA- 
                   
                   
                   
                 0.014 
                 0.000 
                 8.214 
                 0.000 
                 0.050 
                 3.949 
                 0.007 
                 1.807 
               
               
                   
                 type ATPase 
               
               
                 AT5G49110 
                 unknown 
                   
                   
                   
                 0.004 
                 0.001 
                 7.611 
                 0.000 
                 0.037 
                 4.819 
                 0.002 
                 1.562 
               
               
                   
                 protein; 
               
               
                   
                 INVOLVED 
               
               
                   
                 IN: biological 
               
               
                   
                 process 
               
               
                   
                 unknown; 
               
               
                   
                 LOCATED 
               
               
                   
                 IN: cellular 
               
               
                   
                 component 
               
               
                   
                 unknown; 
               
               
                   
                 EXPRESSED 
               
               
                   
                 IN: cultured 
               
               
                   
                 cell; Has 
               
               
                   
                 30201 Blast 
               
               
                   
                 hits to 17322 
               
               
                   
                 proteins in 
               
               
                   
                 780 species: 
               
               
                   
                 Archae - 12; 
               
               
                   
                 Bacteria - 1396; 
               
               
                   
                 Metazoa - 17338; 
               
               
                   
                 Fungi - 3422; 
               
               
                   
                 Plants - 503 
               
               
                   
               
               
                   a According to Cools et al., 2011 
               
               
                   b According to Culligan et al., 2006 
               
               
                   c According to Yoshiyama et al., 2009 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Annotated  Arabidopsis  SIM/SMR genes 
               
             
          
           
               
                   
                 AGI locus 
                 Annotation 
               
               
                   
                   
               
               
                   
                 At5g04470 
                 SIM 
               
               
                   
                 At3g10525 
                 SMR1 
               
               
                   
                 At1g08180 
                 SMR2 
               
               
                   
                 At5g02420 
                 SMR3 
               
               
                   
                 At5g02220 
                 SMR4 
               
               
                   
                 At1g07500 
                 SMR5 
               
               
                   
                 At5g40460 
                 SMR6 
               
               
                   
                 At3g27630 
                 SMR7 
               
               
                   
                 At1g10690 
                 SMR8 
               
               
                   
                 At1g51355 
                 SMR9 
               
               
                   
                 At2g28870 
                 SMR10 
               
               
                   
                 At2g28330 
                 SMR11 
               
               
                   
                 At2g37610 
                 SMR12 
               
               
                   
                 At5g59360 
                 SMR13 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 DNA ploidy level distribution in transgenic plants 
               
               
                 overexpressing SMR4, SMR5, or SMR7 
               
             
          
           
               
                 Ploidy (%) 
                 Col-0 
                 SMR4 OE   
                 SMR5 OE   
                 SMR7 OE   
               
               
                   
               
               
                  2C 
                 19.6 ± 0.2 
                 17.1 ± 0.1 
                 23.6 ± 0.9 
                 24.2 ± 1.3 
               
               
                  4C 
                 26.3 ± 1.2 
                 19.4 ± 0.5 
                 21.3 ± 0.8 
                 29.2 ± 0.7 
               
               
                  8C 
                 49.2 ± 0.5 
                 34.9 ± 3.4 
                 34.8 ± 0.5 
                 36.1 ± 0.2 
               
               
                 16C 
                  4.6 ± 0.7 
                 27.1 ± 3.1 
                 19.6 ± 0.2 
                  9.5 ± 0.9 
               
               
                 32C 
                 0.2 ± 0 
                  1.5 ± 0.6 
                  0.7 ± 0.1 
                  1.1 ± 0.1 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                 List of primers used for cloning, genotyping, and RT-PCR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Promoter cloning primers 
               
             
          
           
               
                 SIAMESE 
                 Fw 
                 ATAGAAAAGTTGGTATTGTAATTATATATGAAAAAATAGTAAT  
               
               
                   
                   
                 (SEQ ID NO: 7) 
               
               
                   
                 Rev 
                 GTACAAACTTGTTCTTTTTTGTTTATATAAATATTAAATGT  
               
               
                   
                   
                 (SEQ ID NO: 8) 
               
               
                 SMR1 
                 Fw 
                 ATAGAAAAGTTGTCACAAGTGCATTTTTAATTTGTAGGA  
               
               
                   
                   
                 (SEQ ID NO: 9) 
               
               
                   
                 Rev 
                 GTACAAACTTGCATCTAAACTTGTGTATGTTTTTGTTTTTTGG  
               
               
                   
                   
                 (SEQ ID NO: 10) 
               
               
                 SMR2 
                 Fw 
                 ATAGAAAAGTTGGTAACTCCTTCGGCATCTTTGT (SEQ ID NO: 11) 
               
               
                   
                 Rev 
                 GTACAAACTTGTGGTCACATGGATGTGAAAGTTT (SEQ ID NO: 12) 
               
               
                 SMR3 
                 Fw 
                 ATAGAAAAGTTGGTATTTTAAATTACGATTTCAAAATCTTGA  
               
               
                   
                   
                 (SEQ ID NO: 13) 
               
               
                   
                 Rev 
                 GTACAAACTTGTTAGACAAGTTTTACAGAGAGAAAGAAGAG  
               
               
                   
                   
                 (SEQ ID NO: 14) 
               
               
                 SMR4 
                 Fw 
                 ATAGAAAAGTTGGTGAAACACAAAGCATCTTCG (SEQ ID NO: 15) 
               
               
                   
                 Rev 
                 GTACAAACTTGTTCTTCTCTCTCGAACTCG (SEQ ID NO: 16) 
               
               
                 SMR5 
                 Fw 
                 ATAGAAAAGTTGGTCAGAACGAACAAAAG (SEQ ID NO: 17) 
               
               
                   
                 Rev 
                 GTACAAACTTGTTTTTGTCCGCTCTCTCG (SEQ ID NO: 18) 
               
               
                 SMR6 
                 Fw 
                 ATAGAAAAGTTGGTCAGTGTGTCAAAACCGACG (SEQ ID NO: 19) 
               
               
                   
                 Rev 
                 GTACAAACTTGTCTCTCTTTAACTAACTCAAAACCAAGA  
               
               
                   
                   
                 (SEQ ID NO: 20) 
               
               
                 SMR7 
                 Fw 
                 AGAAAAGTTGCGTTGACGCGGGAAAATTAA (SEQ ID NO: 21) 
               
               
                   
                 Rev 
                 GTACAAACTTGCTTAAAACAGTTGGAGATTGAG (SEQ ID NO: 22) 
               
               
                 SMR8 
                 Fw 
                 ATAGAAAAGTTGGTAGATCCCACATTACTTAAGAAATTGG  
               
               
                   
                   
                 (SEQ ID NO: 23) 
               
               
                   
                 Rev 
                 GTACAAACTTGTGACTTCTCTCGAATGTGAATGAAGA (SEQ ID NO: 24) 
               
               
                 SMR9 
                 Fw 
                 ATAGAAAAGTTGGTACATATAAAGGTGTTATACACACCCTT  
               
               
                   
                   
                 (SEQ ID NO: 25) 
               
               
                   
                 Rev 
                 GTACAAACTTGTTTTTGAGACCAGAATAAGAGAGAAG (SEQ ID NO: 26) 
               
               
                 SMR10 
                 Fw 
                 ATAGAAAAGTTGGTTTTAAAAAACCGTTTCAAACTAGTGC  
               
               
                   
                   
                 (SEQ ID NO: 27) 
               
               
                   
                 Rev 
                 GTACAAACTTGTCTTTGAGAAGAAACGTCGCTC (SEQ ID NO: 28) 
               
               
                 SMR11 
                 Fw 
                 ATAGAAAAGTTGGTTGTGGTAATCTACATGGAATTTGC (SEQ ID NO: 29) 
               
               
                   
                 Rev 
                 GTACAAACTTGTTTGGATTCACGAGATCTAAGCA (SEQ ID NO: 30) 
               
               
                 SMR12 
                 Fw 
                 ATAGAAAAGTTGGTTCGGCTCACCTTGTTTTCC (SEQ ID NO: 31) 
               
               
                   
                 Rev 
                 GTACAAACTTGTGTGCGCTTTTTTTTCTTCTCAG (SEQ ID NO: 32) 
               
               
                 SMR13 
                 Fw 
                 ATAGAAAAGTTGGTAAAACTCAAGACACTTCTTTTTTTGG  
               
               
                   
                   
                 (SEQ ID NO: 33) 
               
               
                   
                 Rev 
                 GTACAAACTTGTCTTATCACAAACAGGAAAAGAGAGAGT  
               
               
                   
                   
                 (SEQ ID NO: 34) 
               
               
                   
               
             
          
           
               
                 ORF cloning primers 
               
             
          
           
               
                 SMR4 
                 Fw 
                 AAAAAGCAGGCTTCATGGAGGTGG TGGAGAGGAA G (SEQ ID NO: 35) 
               
               
                   
                 Rev + stop code 
                 AGAAAGCTGGGTCCTAAGCGCAAGCTTCTCTTC (SEQ ID NO: 36) 
               
               
                   
                 Rev - stop code 
                 AGAAAGCTGGGTCAGCGCAAGCTTCTCTTC (SEQ ID NO: 37) 
               
               
                 SMR5 
                 Fw 
                 AAAAAGCAGGCTTCATGGAGGAGAAAAACTACGACG (SEQ ID NO: 38) 
               
               
                   
                 Rev + stop code 
                 AGAAAGCTGGGTCCTAGGTTGCCGCTTGGG (SEQ ID NO: 39) 
               
               
                   
                 Rev - stop code 
                 AGAAAGCTGGGTCGGTTGCCGCTTGGGA (SEQ ID NO: 40) 
               
               
                 SMR7 
                 Fw 
                 AAAAAGCAGGCTTCATGGGAATTTCGAAAAAATCTC (SEQ ID NO: 41) 
               
               
                   
                 Rev + stop code 
                 AGAAAGCTGGGTCTTAACGGCGTTGTATAAACACC (SEQ ID NO: 42) 
               
               
                   
                 Rev - stop code 
                 AGAAAGCTGGGTCACGGCGTTGTATAAACACCA (SEQ ID NO: 43) 
               
               
                   
               
             
          
           
               
                 T-DNA genotyping primers 
               
             
          
           
               
                 SMR5 
                 SALK_100918 
                 LB 
                 GAACGAACAAAAGTGAGCTCG (SEQ ID NO: 44) 
               
               
                   
                   
                 RB 
                 TTTCCCAACCTGACAGAAAAC (SEQ ID NO: 45) 
               
               
                 SMR7 
                 SALK_128496 
                 LB 
                 AAAATCGATAACTAAAACGAACCG (SEQ ID NO: 46) 
               
               
                   
                   
                 RB 
                 AGGCCTTCAATATAGCCCATG (SEQ ID NO: 47) 
               
               
                   
               
             
          
           
               
                 RT-PCR primers 
               
             
          
           
               
                 SIAMESE 
                 Fw 
                 CACAAGATTCCTCCCACCACAG (SEQ ID NO: 48) 
               
               
                   
                 Rev 
                 CAGAGGAGAAGAACCGCTCGAT (SEQ ID NO: 49) 
               
               
                 SMR1 
                 Fw 
                 CACCCACATCCCAAGAACACAAG (SEQ ID NO: 50) 
               
               
                   
                 Rev 
                 GACGGAGGAGAAGAAACGGTCAA (SEQ ID NO: 51) 
               
               
                 SMR2 
                 Fw 
                 AGAGCAGAAACCCAGAAGCCAAG (SEQ ID NO: 52) 
               
               
                   
                 Rev 
                 GAAATCTCACGCGGTCGCTTTCTT (SEQ ID NO: 53) 
               
               
                 SMR3 
                 Fw 
                 CGATCACAAGATTCCGGAGGTG (SEQ ID NO: 54) 
               
               
                   
                 Rev 
                 CGGCTCAGATCAATCGGTATGC (SEQ ID NO: 55) 
               
               
                 SMR4 
                 Fw 
                 GCCGAGAAGCACGATGTATAG (SEQ ID NO: 56) 
               
               
                   
                 Rev 
                 AGATCTGGTGGCTGAAAGTACC (SEQ ID NO: 57) 
               
               
                 SMR5 
                 Fw 
                 AAACTACGACGACGGAGATACG (SEQ ID NO: 58) 
               
               
                   
                 Rev 
                 GCTACCACCGAGAAGAACAAGT (SEQ ID NO: 59) 
               
               
                 SMR6 
                 Fw 
                 GGGCTTCGTTGAAACCAGTCAAG (SEQ ID NO: 60) 
               
               
                   
                 Rev 
                 TTTCTCGGTGCTGGTGGACATTC (SEQ ID NO: 61) 
               
               
                 SMR7 
                 Fw 
                 GCCAAAACATCGATTCGGGCTTC (SEQ ID NO: 62) 
               
               
                   
                 Rev 
                 TCGCCGTGGGAGTGATACAAAT (SEQ ID NO: 63) 
               
               
                 SMR8 
                 Fw 
                 TAACCTATCTCCCGGCGTCACA (SEQ ID NO: 64) 
               
               
                   
                 Rev 
                 GCACTTCAACGACGGTTTACGC (SEQ ID NO: 65) 
               
               
                 SMR9 
                 Fw 
                 GCCACTTCAAGAACCCATCTCC (SEQ ID NO: 66) 
               
               
                   
                 Rev 
                 TCCGGAGTACAACATCCACTCTCT (SEQ ID NO: 67) 
               
               
                 SMR10 
                 Fw 
                 GCAAAGAAGGAGCAACCGTCAAG (SEQ ID NO: 68) 
               
               
                   
                 Rev 
                 CGGTGGACAAATTCTTGGCATCG (SEQ ID NO: 69) 
               
               
                 SMR11 
                 Fw 
                 CTGCTTCGATCTCGGATTGTGTT (SEQ ID NO: 70) 
               
               
                   
                 Rev 
                 GACGAAGGAGGCGGTGTTTTAC (SEQ ID NO: 71) 
               
               
                 SMR12 
                 Fw 
                 GGTATGTCGGAGACGAGCTTGA (SEQ ID NO: 72) 
               
               
                   
                 Rev 
                 GAGTCGGTGTCTTGAACCCATCA (SEQ ID NO: 73) 
               
               
                 SMR13 
                 Fw 
                 GAACCACCAACACCGACAACAAG (SEQ ID NO: 74) 
               
               
                   
                 Rev 
                 GTTCGAGTTTCTCGGCGTCTCT (SEQ ID NO: 75) 
               
               
                 Actin2 
                 Fw 
                 GGCTCCTCTTAACCCAAAGGC (SEQ ID NO: 76) 
               
               
                   
                 Rev 
                 CACACCATCACCAGAATCCAGC (SEQ ID NO: 77) 
               
               
                 EMB2386 
                 Fw 
                 CTCTCGTTCCAGAGCTCGCAAAA (SEQ ID NO: 78) 
               
               
                   
                 Rev 
                 AAGAACACGCATCCTACGCATCC (SEQ ID NO: 79) 
               
               
                 PAC1 
                 Fw 
                 TCTCTTTGCAGGATGGGACAAGC (SEQ ID NO: 80) 
               
               
                   
                 Rev 
                 AGACTGAGCCGCCTGATTGTTTG (SEQ ID NO: 81) 
               
               
                 RPS26C 
                 Fw 
                 GACTTTCAAGCGCAGGAATGGTG (SEQ ID NO: 82) 
               
               
                   
                 Rev 
                 CCTTGTCCTTGGGGCAACACTTT (SEQ ID NO: 83) 
               
               
                   
               
             
          
         
       
     
       REFERENCES 
       [0000]    
       
         Abraham, R. T. (2001). Cell cycle checkpoint signaling through the ATM and ATR kinases.  Genes Dev.  15:2177-2196. 
         Adachi, S., K. Minamisawa, Y. Okushima, S. Inagaki, K. Yoshiyama, Y. Kondou, E. Kaminuma, 
         M. Kawashima, T. Toyoda, M. Matsui, D. Kurihara, S. Matsunaga, and M. Umeda (2011). Programmed induction of endoreduplication by DNA double-strand breaks in  Arabidopsis. Proc. Natl. Acad. Sci. USA  108:10004-10009. 
         Amor, Y., E. Babiychuk, D. Inzé, and A. Levine (1998). The involvement of poly(ADP-ribose) polymerase in the oxidative stress responses in plants.  FEBS Lett.  440:1-7. 
         Bartek, J., and J. Lukas (2001). Pathways governing G1/S transition and their response to DNA damage.  FEBS Lett.  490:117-122. 
         Beeckman, T., and G. Engler (1994). An easy technique for the clearing of histochemically stained plant tissue.  Plant Mol. Biol. Rep.  12:37-42. 
         Beers, R. F., Jr., and I. W. Sizer (1952). A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase.  J. Biol. Chem.  195:133-140. 
         Boudolf, V., K. Vlieghe, G. T. S. Beemster, Z. Magyar, J. A. Torres Acosta, S. Maes, E. Van Der Schueren, D. Inzé, and L. De Veylder (2004). The plant-specific cyclin-dependent kinase CDKB1;1 and transcription factor E2Fa-DPa control the balance of mitotically dividing and endoreduplicating cells in  Arabidopsis. Plant Cell  16:2683-2692. 
         Boudolf, V., T. Lammens, J. Boruc, J. Van Leene, H. Van Den Daele, S. Maes, G. Van Isterdael, E. Russinova, E. Kondorosi, E. Witters, G. De Jaeger, D. Inzé, and L. De Veylder (2009). CDKB1;1 forms a functional complex with CYCA2;3 to suppress endocycle onset.  Plant Physiol.  150:1482-1493. 
         Cadet, J., T. Douki, J.-L. Ravanat, and J. R. Wagner (2012). Measurement of oxidatively generated base damage to nucleic acids in cells: facts and artifacts.  Bioanal. Rev.  4:55-74. 
         Chaturvedi, P., W. K. Eng, Y. Zhu, M. R. Mattern, R. Mishra, M. R. Hurle, X. Zhang, R. S. Annan, Q. Lu, L. F. Faucette, G. F. Scott, X. Li, S. A. Canrr, R. K. Johnson, J. D. Winkler, and B. B. Zhou (1999). Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway.  Oncogene  18:4047-4054. 
         Che, F.-S., Y. Nakajima, N. Tanaka, M. Iwano, T. Yoshida, S. Takayama, I. Kadota, and A. Isogai (2000). Flagellin from an incompatible strain of  Pseudomonas avenae  induces a resistance response in cultured rice cells.  J. Biol. Chem.  275:32347-32356. 
         Chen, G. X., and K. Asada (1990). Hydroxyurea and p-aminophenol are the suicide inhibitors of ascorbate peroxidase.  J. Biol. Chem.  265:2775-2781. 
         Chen, Y., and Y. Sanchez (2004). Chk1 in the DNA damage response: conserved roles from yeasts to mammals.  DNA Repair  3:1025-1032. 
         Churchman, M. L., M. L. Brown, N. Kato, V. Kirik, M. Hillskamp, D. Inzé, L. De Veylder, J. D. Walker, Z. Zheng, D. G. Oppenheimer, T. Gwin, J. Churchman, and J. C. Larkin (2006). SIAMESE, a plant-specific cell cycle regulator, controls endoreplication onset in  Arabidopsis thaliana. Plant Cell  18:3145-3157. 
         Clough, S. J., and A. F. Bent (1998). Floral dip: a simplified method for  Agrobacterium -mediated transformation of  Arabidopsis thaliana. Plant J.  16:735-743. 
         Cools, T., A. Iantcheva, S. Maes, H. Van den Daele, and L. De Veylder (2010). A replication stress-induced synchronization method for  Arabidopsis thaliana  root meristems.  Plant J.  64:705-714. 
         Cools, T., A. Iantcheva, A. K. Weimer, S. Boens, N. Takahashi, S. Maes, H. Van den Daele, G. Van Isterdael, A. Schnittger, and L. De Veylder (2011). The  Arabidopsis thaliana  checkpoint kinase WEE1 protects against premature vascular differentiation during replication stress.  Plant Cell  23:1435-1448. 
         Culligan, K., A. Tissier, and A. Britt (2004). ATR regulates a G2-phase cell-cycle checkpoint in  Arabidopsis thaliana. Plant Cell  16:1091-1104. 
         Culligan, K. M., C. E. Robertson, J. Foreman, P. Doerner, and A. B. Britt (2006). ATR and ATM play both distinct and additive roles in response to ionizing radiation.  Plant J.  48:947-961. 
         De Clercq, A., and D. Inzé (2006). Cyclin-dependent kinase inhibitors in yeast, animals, and plants: a functional comparison.  Crit. Rev. Biochem. Mol. Biol.  41:293-313. 
         De Schutter, K., J. Joubes, T. Cools, A. Verkest, F. Corellou, E. Babiychuk, E. Van Der Schueren, T. Beeckman, S. Kushnir, D. Inzé, and L. De Veylder (2007).  Arabidopsis  WEE1 kinase controls cell cycle arrest in response to activation of the DNA integrity checkpoint.  Plant Cell  19:211-225. 
         De Veylder, L., G. Segers, N. Glab, P. Casteels, M. Van Montagu, and D. Inzé (1997). The  Arabidopsis  Cks1At protein binds the cyclin-dependent kinases Cdc2aAt and Cdc2bAt.  FEBS Lett.  412:446-452. 
         De Veylder, L., T. Beeckman, G. T. S. Beemster, L. Krols, F. Terms, I. Landrieu, E. Van Der Schueren, S. Maes, M. Naudts, and D. Inzé (2001). Functional analysis of cyclin-dependent kinase inhibitors of  Arabidopsis. Plant Cell  13:1653-1667. 
         DePaoli, H. C., G. H. Goldman, and M.-H. S. Goldman (2012). SCI1, the first member of the tissue-specific inhibitors of CDK (TIC) class, is probably connected to the auxin signaling pathway.  Plant Signal. Behav.  7:53-58. 
         DePaoli, H. C., M. S. Brito, A. C. Quiapim, S. P. Teixeira, G. H. Goldman, M. C. Dornelas, and M. H. S. Goldman (2011). Stigma/style cell cycle inhibitor 1 (SCI1), a tissue-specific cell cycle regulator that controls upper pistil development.  New Phytol.  190:882-895. 
         Dewitte, W., and J. A. H. Murray (2003). The plant cell cycle.  Annu. Rev. Plant Biol.  54:235-264. 
         Dissmeyer, N., A. K. Weimer, S. Pusch, K. De Schutter, C. L. Alvim Kamei, M. K. Nowack, B. Novak, G.-L. Duan, Y.-G. Zhu, L. De Veylder, and A. Schnittger (2009). Control of cell proliferation, organ growth, and DNA damage response operate independently of dephosphorylation of the  Arabidopsis  Cdk1 homolog CDKA;1 . Plant Cell  21:3641-3654. 
         Dizdaroglu, M., P. Jaruga, M. Birincioglu, and H. Rodriguez (2002). Free radical-induced damage to DNA: mechanisms and measurement.  Free Radic. Biol. Med.  32:1102-1115. 
         Dubacq, C., A. Chevalier, R. Courbeyrette, C. Petat, X. Gidrol, and C. Mann (2006). Role of the iron mobilization and oxidative stress regulons in the genomic response of yeast to hydroxyurea.  Mol. Genet. Genomics  275:114-124. 
         Dunand, C., M. Crevecoeur, and C. Penel (2007). Distribution of superoxide and hydrogen peroxide in  Arabidopsis  root and their influence on root development: possible interaction with peroxidases.  New Phytol.  174:332-341. 
         Francis, D. (2011). A commentary on the G2/M transition of the plant cell cycle.  Ann. Bot.  107:1065-1070. 
         French, A. P., M. H. Wilson, K. Kenobi, D. Dietrich, U. VoB, S. Ubeda-Tomas, T. P. Pridmore, and D. M. Wells (2012). Identifying biological landmarks using a novel cell measuring image analysis tool: Cell-o-Tape.  Plant Methods  8:7. 
         Gaj, T., C. A. Gersbach, and C. F. BARBAS III (2013). ZFN, TALEN and CRISPR/Cas-based methods for genome engineering.  Trends Biotechnol.  31:397-405. 
         Galbraith, D. W., K. R. Harkins, and S. Knapp (1991). Systemic Endopolyploidy in  Arabidopsis thaliana. Plant Physiol.  96:985-989. 
         Garcia, V., H. Bruchet, D. Camescasse, F. Granier, D. Bouchez, and A. Tissier (2003). AtATM is essential for meiosis and the somatic response to DNA damage in plants.  Plant Cell  15:119-132. 
         Gendrel, A.-V., Z. Lippman, R. Martienssen, and V. Colot (2005). Profiling histone modification patterns in plants using genomic tiling microarrays.  Nat. Methods  2:213-218. 
         Guo, Z., S. Kozlov, M. F. Lavin, M. D. Person, and T. T. Paull (2010). ATM activation by oxidative stress.  Science  330:517-521. 
         Hruz, T., O. Laule, G. Szabo, F. Wessendorp, S. Bleuler, L. Oertle, P. Widmayer, W. Gruissem, and P. Zimmermann (2008). GENEVESTIGATOR® V3: a reference expression database for the meta-analysis of transcriptomes.  Adv. Bioinformatics  2008:420747. 
         Irizarry, R. A., B. Hobbs, F. Collin, Y. D. Beazer-Barclay, K. J. Antonellis, U. Scherf, and T. P. Speed (2003). Exploration, normalization, and summaries of high density oligonucleotide array probe level data.  Biostatistics  4:249-264. 
         Juul, T., A. Malolepszy, K. Dybkær, R. Kidmose, J. T. Rasmussen, G. R. Andersen, H. E. Johnsen, J.-E. Jorgensen, and S. U. Andersen (2010). The in vivo toxicity of hydroxyurea depends on its direct target catalase.  J. Biol. Chem.  285:21411-21415. 
         Karimi, M., A. Bleys, R. Vanderhaeghen, and P. Hilson (2007). Building blocks for plant gene assembly.  Plant Physiol.  145:1183-1191. 
         Karimi, M., D. Inzé, and A. Depicker (2002). GATEWAY™ vectors for  Agrobacterium -mediated plant transformation.  Trends Plant Sci.  7:193-195. 
         Kinoshita, E., E. Kinoshita-Kikuta, K. Takiyama, and T. Koike (2006). Phosphate-binding tag, a new tool to visualize phosphorylated proteins.  Mol. Cell. Proteomics  5:749-757. 
         Kurz, E. U., and S. P. Lees-Miller (2004). DNA damage-induced activation of ATM and ATM-dependent signaling pathways.  DNA Repair  3:889-900. 
         Lee, M. G., and P. Nurse (1987). Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2 . Nature  327:31-35. 
         Minami, E., K. Kuchitsu, D.-Y. He, H. Kouchi, N. Midoh, Y. Ohtsuki, and H. Shibuya (1996). Two novel genes rapidly and transiently activated in suspension-cultured rice cells by treatment with N-acetylchitoheptaose, a biotic elicitor for phytoalexin production.  Plant Cell Physiol.  37:563-567. 
         Mittler, R., S. Vanderauwera, M. Gollery, and F. Van Breusegem (2004). Reactive oxygen gene network of plants.  Trends Plant Sci.  9:490-498. 
         Mittler, R., S. Vanderauwera, N. Suzuki, G. Miller, V. B. Tognetti, K. Vandepoele, M. Gollery, V. Shulaev, and F. Van Breusegem (2011).  ROS signaling: the new wave? Trends Plant Sci.  16:300-309. 
         Murashige, T., and F. Skoog (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures.  Physiol. Plant.  15:473-497. 
         Murata, M., K. Fukushima, T. Takao, H. Seki, S. Takeda, and N. Wake (2013). Oxidative stress produced by xanthine oxidase induces apoptosis in human extravillous trophoblast cells.  J. Reprod. Dev.  59:7-13. 
         Norbury, C., and P. Nurse (1992). Animal cell cycles and their control.  Annu. Rev. Biochem.  61:441-468. 
         Nowack, M. K., A. Ungru, K. N. Bjerkan, P. E. Grini, and A. Schnittger (2010). Reproductive cross-talk: seed development in flowering plants.  Biochem. Soc. Trans.  38:604-612. 
         Nowack, M. K., H. Harashima, N. Dissmeyer, X. Zhao, D. Bouyer, A. K. Weimer, F. De Winter, F. Yang, and A. Schnittger (2012). Genetic framework of cyclin-dependent kinase function in  Arabidopsis. Dev. Cell  22:1030-1040. 
         Nyholm, S., G. J. Mann, A. G. Johansson, R. J. Bergeron, A. Grislund, and L. Thelander (1993). Role of ribonucleotide reductase in inhibition of mammalian cell growth by potent iron chelators.  J. Biol. Chem.  268:26200-26205. 
         Peres, A., M. L. Churchman, S. Hariharan, K. Himanen, A. Verkest, K. Vandepoele, Z. Magyar, Y. Hatzfeld, E. Van Der Schueren, G. T. S. Beemster, V. Frankard, J. C. Larkin, D. Inzé, and L. De Veylder (2007). Novel plant-specific cyclin-dependent kinase inhibitors induced by biotic and abiotic stresses.  J. Biol. Chem.  282:25588-25596. 
         Preuss, S. B., and A. B. Britt (2003). A DNA-damage-induced cell cycle checkpoint in  Arabidopsis. Genetics  164:323-334. 
         Reichheld, J.-P., T. Vernoux, F. Lardon, M. Van Montagu, and D. Inzé (1999). Specific checkpoints regulate plant cell cycle progression in response to oxidative stress.  Plant J.  17:647-656. 
         Ricaud, L., C. Proux, J.-P. Renou, O. Pichon, S. Fochesato, P. Ortet, and M.-H. Montané (2007). ATM-mediated transcriptional and developmental responses to 7-rays in  Arabidopsis. PLoS ONE  2:e430. 
         Riou-Khamlichi, C., M. Menges, J. M. S. Healy, and J. A. H. Murray (2000). Sugar control of the plant cell cycle: differential regulation of  Arabidopsis  D-type cyclin gene expression.  Mol. Cell. Biol.  20:4513-4521. 
         Roeder, A. H. K., V. Chickarmane, A. Cunha, B. Obara, B. S., Manjunath, and E. M. Meyerowitz (2010). Variability in the control of cell division underlies sepal epidermal patterning in  Arabidopsis thaliana. PLoS Biol.  8:e1000367. 
         Roldan-Arjona, T., and R. R. Ariza (2009). Repair and tolerance of oxidative DNA damage in plants.  Mutat. Res.  681:169-179. 
         Rozan, L. M., and W. S. El-Deiry (2007). p53 downstream target genes and tumor suppression: a classical view in evolution. Cell Death Differ. 14:3-9. 
         Sherr, C. J., and J. M. Roberts (1995). Inhibitors of mammalian G1 cyclin-dependent kinases.  Genes Dev.  9:1149-1163. 
         Shieh, S.-Y., J. Ahn, K. Tamai, Y. Taya, and C. Prives (2000). The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites.  Genes Dev.  14:289-300. 
         Tsukagoshi, H. (2012). Defective root growth triggered by oxidative stress is controlled through the expression of cell cycle-related genes.  Plant Sci.  197:30-39. 
         Tsukagoshi, H., W. Busch, and P. N. Benfey (2010). Transcriptional regulation of ROS controls transition from proliferation to differentiation in the root.  Cell  143:606-616. 
         Tusher, V. G., R. Tibshirani, and G. Chu (2001). Significance analysis of microarrays applied to the ionizing radiation response.  Proc. Natl. Acad. Sci. USA  98:5116-5121. 
         Van Leene, J., J. Hollunder, D. Eeckhout, G. Persiau, E. Van De Slijke, H. Stals, G. Van Isterdael, A. Verkest, S. Neirynck, Y. Buffel, S. De Bodt, S. Maere, K. Laukens, A. Pharazyn, P. C. G. Ferreira, N. Eloy, C. Renne, C. Meyer, J.-D. Faure, J. Steinbrenner, J. Beynon, J. C. Larkin, Y. Van de Peer, P. Hilson, M. Kuiper, L. De Veylder, H. Van Onckelen, D. Inzé, E. Witters, and G. De Jaeger (2010). Targeted interactomics reveals a complex core cell cycle machinery in  Arabidopsis thaliana. Mol. Syst. Biol.  6:397. 
         Vandenabeele, S., S. Vanderauwera, M. Vuylsteke, S. Rombauts, C. Langebartels, H. K. Seidlitz, M. Zabeau, M. Van Montagu, D. Inzé, and F. Van Breusegem (2004). Catalase deficiency drastically affects gene expression induced by high light in  Arabidopsis thaliana. Plant J.  39:45-58. 
         Vanderauwera, S., N. Suzuki, G. Miller, B. van de Cotte, S. Morsa, J.-L. Ravanat, A. Hegie, C. Triantaphylides, V. Shulaev, M. C. E. Van Montagu, F. Van Breusegem, and R. Mittler (2011). Extranuclear protection of chromosomal DNA from oxidative stress.  Proc. Natl. Acad. Sci. USA  108:1711-1716. 
         Vernoux, T., R. C. Wilson, K. A. Seeley, J.-P. Reichheld, S. Muroy, S. Brown, S. C. Maughan, C. S. Cobbett, M. Van Montagu, D. Inzé, M. J. May, and Z. R. Sung (2000). The ROOT MERISTEMLESS1/CADMIUM SENSITIVE2 gene defines a glutathione-dependent pathway involved in initiation and maintenance of cell division during postembryonic root development.  Plant Cell  12:97-109. 
         Wang, H., Y. Zhou, S. Gilmer, S. Whitwill, and L. C. Fowke (2000). Expression of the plant cyclin-dependent kinase inhibitor ICK1 affects cell division, plant growth and morphology.  Plant J.  24:613-623. 
         Wang, H., Q. Qi, P. Schorr, A. J. Cutler, W. L. Crosby, and L. C. Fowke (1998). ICK1, a cyclin-dependent protein kinase inhibitor from  Arabidopsis thaliana  interacts with both Cdc2a and CycD3, and its expression is induced by abscisic acid.  Plant J.  15:501-510. 
         Yoshiyama, K., P. A. Conklin, N. D. Huefner, and A. B. Britt (2009). Suppressor of gamma response 1 (SOG1) encodes a putative transcription factor governing multiple responses to DNA damage.  Proc. Natl. Acad. Sci. USA  106:12843-12848. 
         Yoshiyama, K. O., J. Kobayashi, N. Ogita, M. Ueda, S. Kimura, H. Maki, and M. Umeda (2013). ATM-mediated phosphorylation of SOG1 is essential for the DNA damage response in  Arabidopsis. EMBO Rep.  14:817-822. 
         Zhou, B.-B., and S. J. Elledge (2000). The DNA damage response: putting checkpoints in perspective.  Nature  408:433-439.