Abstract
Reactive oxygen species (ROS) are toxic by-products generated continuously during seed desiccation, storage, and germination, resulting in seed deterioration and therefore decreased seed longevity. The toxicity of ROS is due to their indiscriminate reactivity with almost any constituent of the cell, such as lipids, proteins, and DNA. The damage to the genome induced by ROS has been recognized as an important cause of seed deterioration. A prominent DNA lesion induced by ROS is 7,8-dihydro-8-oxoguanine (8-oxo-G), which can form base pairs with adenine instead of cytosine during DNA replication and leads to GC→TA transversions. In Arabidopsis, AtOGG1 is a DNA glycosylase/apurinic/apyrimidinic (AP) lyase that is involved in base excision repair for eliminating 8-oxo-G from DNA. In this study, the functions of AtOGG1 were elaborated. The transcript of AtOGG1 was detected in seeds, and it was strongly up-regulated during seed desiccation and imbibition. Analysis of transformed Arabidopsis protoplasts demonstrated that AtOGG1–yellow fluorescent protein fusion protein localized to the nucleus. Overexpression of AtOGG1 in Arabidopsis enhanced seed resistance to controlled deterioration treatment. In addition, the content of 8-hydroxy-2′-deoxyguanosine (8-oxo-dG) in transgenic seeds was reduced compared to wild-type seeds, indicating a DNA damage-repair function of AtOGG1 in vivo. Furthermore, transgenic seeds exhibited increased germination ability under abiotic stresses such as methyl viologen, NaCl, mannitol, and high temperatures. Taken together, our results demonstrated that overexpression of AtOGG1 in Arabidopsis enhances seed longevity and abiotic stress tolerance.
Introduction
Reactive oxygen species (ROS) are generated continuously as by-products of aerobic metabolism or as a consequence of exposure to abiotic stresses such as heat, drought, salinity, and redox-active compounds (Tsang et al., 1991; Bowler et al., 1992; Park et al., 1992; Foyer et al., 1994; Alscher et al., 1997). Although recent research has suggested that ROS act as important signalling molecules participating in regulating plant responses to abiotic and biotic stress (Apel and Hirt, 2004; Foyer and Shigeoka, 2011; Maruta et al., 2012). ROS are ubiquitous and highly reactive oxidizing agents that act as an important source of stress for cells. Most macromolecules, including lipids, proteins, and nucleic acids, can be oxidized by ROS, resulting in cell disruption and organism lesions (Pacifici and Davies, 1991; Smirnoff, 1993; Alscher et al., 1997; Mittler, 2002; Nishimura, 2002; Apel and Hirt, 2004). Therefore, oxidative stress induced by ROS has been suggested to be an important causative agent of mutagenesis, aging, and pathogenesis (Farr and Kogoma, 1991; Pacifici and Davies, 1991; Michaels and Miller, 1992; Apel and Hirt, 2004). Furthermore, ROS are often considered to be a main cause of seed deterioration associated with loss of seed vigor and viability (McDonald, 1999; Bailly et al., 2008; El-Maarouf-Bouteau et al., 2011). To eliminate ROS, cells develop a number of ROS scavengers such as superoxide dismutase, peroxidase (Tsang et al., 1991; Bowler et al., 1992; Bailly et al., 1996; Apel and Hirt, 2004), and vitamins (Packer et al., 1979; Sattler et al., 2004). Several studies have reported enhanced seed longevity through elimination of ROS by overaccumulated ROS scavengers in transgenic seeds (Lee et al., 2010; Zhou et al., 2012). In living cells, a major target of ROS is the electron-rich bases of DNA, resulting in a diverse range of genotoxic modifications (Lindahl, 1993; Tuteja et al., 2009). DNA oxidization induced by ROS is thought to be the major source of DNA damage during seed storage and seed germination (Dandoy et al., 1987; Bray and West, 2005). Under normal storage conditions, the loss of seed viability is often associated with the accumulation of DNA breaks and chromosome aberration, establishing a link between DNA damage and reduced germination potential during senescence of the embryo in dry seeds (Cheah and Osborne, 1978; Osborne, 1982; Waterworth et al., 2011).
ROS induce a variety of lesions in DNA, including oxidized bases, apurinic/apyrimidinic (AP) sites and DNA strand breaks (Klungland et al., 1999). Among all the DNA lesions induced by ROS, 7,8-dihydro-8-oxoguanine (8-oxo-G) is a dominant one as a result of ROS-induced hydroxylation of the C-8 position of guanine (Kasai and Nishimura, 1984; Yoshida et al., 2002). During DNA replication, the 8-oxo-G can pair with both cytosine and adenine with almost equal efficiency, giving rise to GC→TA transversions and inducing mutagenesis (Wood et al., 1990; Kouchakdjian et al., 1991; Moriya et al., 1991; Shibutani et al., 1991; Moriya, 1993). In DNA, the 8-oxo-G combines with a deoxyribose in deoxyguanosine, gives rise to 8-hydroxy-2′-deoxyguanosine (8-oxo-dG) (Fig. 1A) (Yoshida et al., 2002). To cope with the damage induced by ROS, cells develop a variety of protective measures including DNA repair enzymes (Ohtsubo et al., 1998; Dany and Tissier, 2001; Garcia-Ortiz et al., 2001; Waterworth et al., 2010) and protein repair enzymes (Petropoulos and Friguet, 2006; Oge et al., 2008). Extensive studies have revealed a base excision repair (BER) system involving excision of 8-oxo-G from DNA to eliminate its mutagenic effects (Fig. 1B) (Britt, 1996; Wood, 1996). First, the DNA N-glycosyl bond is excised by DNA glycosylases to liberate the free 8-oxo-G base and generate an AP site (Michaels and Miller, 1992; Wood, 1996). Then, the AP site is hydrolysed by an AP endonuclease at the phosphodiester bond 5′ or 3′ and retains a nick (Vonarx et al., 1998; McCullough et al., 1999). The completion of repair is mediated by a DNA polymerase and ligase after the terminal sugar-phosphate moiety of the retained nick is removed by an exonuclease (DNA deoxyribophosphodiesterase) (Franklin and Lindahl, 1988; Sandigursky and Franklin, 1992). Studies in Escherichia coli have revealed two DNA glycosylases, MutM (or Fpg) and MutY, which participate in the BER system to eliminate the mutation induced by 8-oxo-G (Boiteux et al., 1990; Tchou et al., 1991; Michaels and Miller, 1992). MutM shows a clear preference for excision of 8-oxo-G from the 8-oxo-G:C to that of 8-oxo-G:A pairs in DNA (Tchou et al., 1991; Boiteux et al., 1992; Castaing et al., 1993). In contrast, MutY recognizes 8-oxo-G:A and catalyses the excision of adenine, providing another pathway to repair DNA damage induced by 8-oxo-G (Michaels and Miller, 1992; Michaels et al., 1992; McGoldrick et al., 1995). Inactivation of either mutM or mutY causes a high frequency of spontaneous GC→TA mutations, and a mutY mutM double mutant has a 20-fold higher mutation rate than either mutator alone (Michaels et al., 1992). In eukaryotes, the functional homologue of MutM was first identified from Saccharomyces cerevisiae by complementation of the phenotype of mutY mutM double mutant and designated OGG1 (Nash et al., 1996; van der Kemp et al., 1996). Since then, human and other mammal OGG1 homologues have also been isolated and characterized (Arai et al., 1997; Radicella and Boiteux, 1997; Radicella et al., 1997; Roldan-Arjona et al., 1997; Rosenquist et al., 1997).
Schematic diagram of 8-oxo-dG formation and enzymatic repair by the base excision repair (BER) pathway. (A) The guanine of 2'-deoxyguanosine in DNA is hydroxylated by ROS and generates 8-hydroxy-2'-deoxyguanosine (8-oxo-dG), which has two isomers. The grey rectangle indicates the 8-oxo-G in 8-oxo-dG. The grey arrows point to the 8-position carbon atom of guanine. (B) The BER pathway for the repair of 8-oxo-G-mediated mispairs in DNA. The grey arrow indicates the enzyme reaction position.
The OGG1 protein is a bifunctional DNA glycosylase/AP lyase which excises 8-oxo-G and cleaves DNA at the 3′-side of the resulting AP site via a β-elimination reaction (Girard et al., 1997; Sandigursky et al., 1997). Both yeast OGG1 (yOGG1) and human OGG1 (hOGG1) proteins display a preference for 8-oxo-G excision from DNA duplexes containing 8-oxo-G:C, and the cleavage efficiencies are 8-oxo-G:C > 8-oxo-G:T >> 8-oxo-G:G and 8-oxo-G:A (Girard et al., 1997; Rosenquist et al., 1997). The hOGG1 is expressed as four splice variants (types 1a, 1b, 1c, and 2) by alternative splicing, leading to different intracellular localization (Aburatani et al., 1997; Takao et al., 1998). The three tagged isoforms, types 1b, 1c, and 2, are localized in the mitochondria, while type 1a is mainly found in the nucleus and to a lesser extent in the mitochondria (Takao et al., 1998; Nishioka et al., 1999). Expression of hOGG1 in a DNA repair-deficient E. coli mutM mutY strain or a yOGG1 mutant partially suppresses the spontaneous mutation phenotype (Radicella et al., 1997; Roldan-Arjona et al., 1997). The hOGG1 protein is involved in many diseases such as carcinogenesis and aging, and can be inhibited by NO (Jaiswal et al., 2001; Osterod et al., 2001; Shinmura and Yokota, 2001). In mice, OGG1 has an important role in repairing genomic damage caused by oxidative stress under ischemic conditions thereby protecting neurons from damage by ROS (Liu et al., 2011). Further studies in Eker rats revealed that deficiency in tuberin is associated with reduced expression of OGG1 and the accumulation of significant levels of 8-oxo-dG, implying that OGG1 may be regulated by tuberin (Habib et al., 2010).
The functional homologue of hOGG1 was also identified in Arabidopsis (Dany and Tissier, 2001; Garcia-Ortiz et al., 2001). Arabidopsis thaliana OGG1 (AtOGG1) is also a bifunctional DNA glycosylase/AP lyase with significant sequence identity to yeast and human OGG1 proteins (Garcia-Ortiz et al., 2001). In contrast to the hOGG1, AtOGG1 does not appear to undergo alternative splicing since there is only one isoform (Dany and Tissier, 2001). It has been reported that AtOGG1 contains a putative nucleus-localization signal peptide (Garcia-Ortiz et al., 2001), but the subcellular localization of AtOGG1 remains to be clearly established. In vitro experiments indicate that AtOGG1 prefers to cleave 8-oxo-G:C mispair in duplex DNA (Morales-Ruiz et al., 2003) to form a Schiff base with 8-oxo-G in the presence of NaBH4 (Garcia-Ortiz et al., 2001), and is a feature of all bifunctional DNA glycosylases/AP lyases. Furthermore, expression of AtOGG1 in an E. coli strain deficient in 8-oxo-G repair can partially revert its spontaneous mutant phenotype (Dany and Tissier, 2001; Garcia-Ortiz et al., 2001). Comparing the in vitro substrate specificities of AtOGG1 and AtMMH in vitro revealed that AtOGG1 was more active in excising 8-oxo-G from an oligonucleotide while AtMMH appeared to preferentially recognize depurinated DNA as a substrate, suggesting that the two enzymes have been retained in plants during evolution for their specialized activities (Murphy and George, 2005). The transcripts of AtOGG1 were found in a variety of plant tissues including roots, stems, leaves, and flowers (Garcia-Ortiz et al., 2001).
Compared to bacteria, yeast, and mammals, the physiological function of plant OGG1 is still unclear. In this study we show that AtOGG1 was highly expressed in developing and imbibing seeds and that AtOGG1–yellow fluorescent protein (YFP) fusion protein was localized in the nucleus. Overexpression of AtOGG1 in Arabidopsis seeds conferred enhanced seed resistance to controlled deterioration treatment (CDT). In addition, we found that the amount of 8-oxo-dG was remarkably low in transgenic seeds compared to wild-type seeds with or without CDT, indicating a significant role for AtOGG1 in seed longevity. Furthermore, transgenic seeds also displayed improved germination performance under abiotic stresses including high temperatures, high salinity, osmotic stresses, and especially under oxidative stresses imposed by methyl viologen (MV).
Materials and methods
Plant materials and growth conditions
A. thaliana (ecotype Columbia-0) plants were grown routinely in a greenhouse under 22±1 °C with a light/dark regime of 16 h light/8 h dark. Sterilized seeds were sown on Petri dishes containing half-strength Murashige and Skoog (MS) medium (Duchefa, Haarlem, The Netherlands) and then stratified at 4 °C for 2 days. After stratification, seeds were germinated under the same growth conditions as above and seedlings were transferred to soil after 2 weeks. In all experiments seeds were harvested from wild-type and transgenic plants grown under identical conditions.
Cloning of AtOGG1 and generation of AtOGG1 overexpression and RNA-interference-silenced lines
The full-length cDNA sequence of AtOGG1 was obtained from GenBank (accession no. NM_102020). The primer pair 5′-ACGGCGATGAAGAGACCTC-3′ and 5′-GATTCCTCGTAGCTTAGGTAGAC-3′ was used for gene cloning. Using cDNAs generated from Arabidopsis leaves, a fragment containing the open reading frame (ORF) of AtOGG1 was amplified by PCR and subcloned into the pGEM T-Easy vector (Promega, Madison, WI, USA).
To construct the plant-transforming vector, the ORF of AtOGG1 was amplified using the primer pair 5′-TCCCCCGGGATGAAGAGACCTCGACCTAC-3′ and 5′-CGAGCTCTCATGGCTTCAACGTATCAC-3′. The PCR products were digested by SmaI and SacI (indicated by underlining in the forward and reverse primers above) and subcloned into the binary plasmid pBI121 (Clontech, Palo Alto, CA, USA) by replacing the β-glucuronidase (GUS) gene, to generate the plant-transforming vector pBI121-AtOGG1. The plant-transformation vector containing AtOGG1 under the control of the cauliflower mosaic virus 35S promoter was electroporated into Agrobacterium tumefaciens strain EHA105. Transformation of A. thaliana was conducted by the floral dip method (Clough and Bent, 1998). T0 seeds were harvested and germinated on a sterile medium containing 50 μg ml−1 kanamycin to select the transformants. The heterozygous transgenic plants were further characterized by a 3:1 segregation with respect to kanamycin resistance. Similarly, T3 homozygous seeds were obtained and confirmed by resistance.
To obtain AtOGG1-silenced lines, RNA interference (RNAi) vector pFGC5941 (TAIR) was used for generation of the silencing constructs. A 378 bp region of AtOGG1 was amplified by PCR to generate inverted repeat products using two primer pairs: 5′-CATGCCATGGATGGATGAAGAGACCTCGAC-3′ and 5′-GGGATTTAAATGAAATCAGACCATAGCTCAG-3′ (the first pair, forward and reverse), and 5′-TCCCCCGGGATGGATGAAGAGACCTCGAC-3′ and 5′-CGGGATCCGAAATCAGACCATAGCTCAG-3′ (the second pair, forward and reverse). The products were subcloned into pFGC5941 in a two-step cloning procedure that orients the fragments as inverted repeats separated by an intron from Petunia hybrida chalcone synthase A gene. The RNAi construct was then electroporated into strain EHA105 and used to transform Arabidopsis as described above. Transformed plants were selected by basta resistance. The transcript and protein levels of AtOGG1 in basta-resistant plants were determined by real-time PCR and Western blotting respectively.
Subcellular localization assay
To investigate the subcellular location of AtOGG1, vector pA7-YFP, a pre-made vector in pUC18 containing a YFP gene (Voelker et al., 2006), was used to study transient gene expression. Using the primer pair 5′-GCGTCGACATGAAGAGACCTCGACCTAC-3′ and 5′-TCCCCCGGGTGGCTTCAACGTATCAC-3′, SalI and SmaI sites (indicated by underlining in the primer pair above) were introduced into the ORF of AtOGG1 and the resulting DNA fragment was cloned into pA7-YFP via the SalI and SmaI sites to generate the vector AtOGG1–YFP. The recombinant construct was electroporated into protoplasts of Arabidopsis suspension-cultured cells. For the colocalization assay, Arabidopsis protoplasts were co-electroporated with AtOGG1–YFP, and the DNA of a single organelle marker as indicated. Mitotracker (Invitrogen, Carlsbad, CA, USA) was used to stain mitochondria. The subcellular locations of fusion proteins were examined by confocal laser scanning microscopy 12–16 h after electroporation. Transient expression analysis was carried out essentially as previously described (Miao and Jiang, 2007).
RNA extraction and real-time PCR
Total RNA was extracted from dry mature and imbibing seeds of transgenic and wild-type plants using the Universal Plant RNA Extraction Kit (BioTeke, Beijing, China). Purified RNA was digested with RNase-free DNase I (Takara, Dalian, China) to eliminate DNA contamination. RNA quality and quantity were determined by electrophoresis and spectrophotometry. First-strand cDNA was synthesized from the total RNA with the PrimeScript 1st Strand cDNA Synthesis Kit (Takara). Real-time PCR was performed on an IQ5 Multicolor Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) using the SYBR Green Real-time PCR Master Mix (Toyobo, Shanghai, China). AtActin2 was used as an internal reference. Gene-specific primers (forward and reverse, respectively): for AtOGG1 were 5′-TACAGAGCCAAATACATAACAG-3′ and 5′-TGCTACCTTCGGACCAAC-3′; for AtMMH (AT1G52500) were 5′-AGCCAGAATCCACCCGTT-3′ and 5′-GTCCTTCCACCAGCAGTAATG-3′; for AtARP (AT2G41460) were 5′-TATCAACAACAGCAAGCGAA-3′ and 5′-TTCTTGAACAGTCTCGCCTC-3′; for AtLIG1 (AT1G08130) were 5′-GCGGTTAGGGTTCTCAGGT-3′ and 5′-TCCACACACCGCCACTTAG-3′; for AtPARP2 (AT2G31320) were 5′-CGTATTCTGCGTCCTGTATTGT-3′ and 5′-CGTCTCTGATATCTGTCAGTCCAC-3′; for AtRAD51 (AT5G20850) were 5′-TGAGGGAACATTCAGGCCAC-3′ and 5′-AGAGAGCGGTAGCACTATCG-3′; and for AtActin2 (AT3G18780) were 5′-ATTACCCGATGGGCAAGTCA-3′ and 5′-TGCTCATACGGTCAGCGATA-3′. The conditions for real-time PCR were as follows: 95 °C for 5 min, 40 thermal cycles of 95 °C for 10 s, 58 °C for 10 s, and 72 °C for 20 s, followed by 75 thermal cycles with ramping at a rate of 0.5 °C between 58 and 95 °C to check fusion curves and verify the specificity of the PCR amplification. The amount of cDNA was calculated using the comparative CT method (Schmittgen and Livak, 2008) using Bio-Rad iQ5 2.0 Standard Edition Optical System software. Data represent three biological replicates each consisting of three technical replicates.
CDT
CDT was conducted according to Tesnier et al. (2002) and Oge et al. (2008) with minor modifications. Dry mature seeds were harvested and stored at 4 °C for 15 days before CDT. Each step of CDT was performed in airtight tube carriers containing appropriate saturated solution of salts to obtain stable relative humidity (RH). The carriers were placed in a dark incubator at appropriate temperature for various numbers of days. Temperature and RH were monitored with a Testo 610 controller placed inside the tube carriers. Seeds were placed in microcentrifuge tubes with lids removed and equilibrated for 3 days at 85% RH (15 °C) in the presence of an appropriate saturated solution of KCl. Untreated controls were immediately dried for 3 days at 33% RH (20 °C) in the presence of an appropriate saturated solution of MgCl2. For the controlled treatment, a saturated solution of KCl was used to obtain 82% RH at 40 °C to equilibrate the seeds to 15–20% moisture content. Seeds were treated under these storage conditions for 1–7 days. After that, seeds were dried for 3 days at 33% RH (20 °C). Then seeds were surface sterilized and stored at 4 °C for 2 days before sowing. Four sets of 100 seeds were used for each genotype and germination was monitored with a microscope every day and radicle protrusion of the seeds was scored until a plateau was reached, indicating completion of seed germination. Cotyledon expansion was scored 10 days after sowing and was expressed as a percentage of the seeds that had germinated. Seed moisture content was measured by weighing the seeds before and after drying at 105 °C for 24 h, and relative moisture content was expressed as percentage fresh weight for each RH. This study was replicated for three times and similar results were obtained.
Abiotic stress treatments
For chemical-induced stress treatments, four replicates of 100 dry mature seeds of each genotype were germinated in Petri dishes containing half-strength MS medium with 5–200 μM MV and 75–200 mM NaCl, or 150–600 mM mannitol (Man). The thermotolerance assay was conducted according to (Yokotani et al., 2008) with minor modifications. Briefly, four replicates of 100 dry mature seeds of each genotype were placed in microcentrifuge tubes with the lid removed and equilibrated for 3 days at 85% RH (15 °C) before heat stress. After equilibration, the tubes were first immersed in water at 50, 51, and 52 °C for 30 min, and then immediately dipped in water at room temperature to eliminate the heat stress. Seeds were surface sterilized and sown on half-strength MS medium. Germination and cotyledon expansion percentages were obtained as described for CDT.
DNA extraction and quantification of 8-oxo-dG
Total genomic DNA was extracted from seeds of each genotype with or without CDT using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Purified DNA was digested with proteinase K (10 mg ml−1, Sigma-Aldrich) for 2 h at 37 °C according to the manufacturer’s instructions and subsequently deproteinized using chloroform/isoamyl alcohol. DNA (5 μg) was hydrolysed by incubating with S1 nuclease (10 units; Takara) for 3 h at 37 °C followed by treatment with alkaline phosphatase (10 units; Takara) for 2 h at 37 °C. Free deoxynucleosides were isolated by filtering the whole reaction mixture using an Amicon Ultra-0.5 (Millipore, Billerica, MA, USA) filtration unit. Detection and quantification of 8-oxo-dG in the purified deoxynucleosides were carried out using the DNA Damage ELISA Kit (Stressgen, Enzo, San Diego, CA, USA) according to the manufacturer’s instructions.
Protein expression and Western blotting
The full-length cDNA of AtOGG1 was cloned into the pET14b vector (Novagen, San Diego, CA, USA) and expressed in E. coli strain BL21 (DE3) as described by Chu et al. (2011). The recombinant proteins were purified following the manufacturer’s protocol (Novagen). Polyclonal antiserum was raised in mouse and used in Western blotting. Western blot analysis was performed as described by Chu et al. (2011) with minor modifications. Protein samples were separated by SDS/PAGE and the gels were transferred onto nitrocellulose membranes (Schleicher and Schuell, Keene, NH, USA). The primary antibody was the polyclonal antiserum described above at 1:2000 dilution, and the secondary antibody was goat anti-mouse immunoglobulin horseradish peroxidase antibody (Sigma-Aldrich, St Louis, MO, USA) at 1:5000 dilution. ECL-Plus Western Blotting Detection Reagents (Invitrogen) were used for detection, according to the supplier’s instructions.
Tetrazolium assay
Seed viability was estimated using tetrazolium staining. Arabidopsis seeds were incubated in a 1% (w/v) aqueous solution of 2,3,5-triphenyltetrazolium chloride (Alfa, Ward Hill, MA, USA) at 30 °C in darkness for 2 days as described by Wharton (1955). Tetrazolium salts were metabolically reduced to highly coloured end products called formazans by NADH-dependent reductases of the endoplasmic reticulum.
Results
Expression analysis of AtOGG1 during seed development and germination
A previous study has indicated that AtOGG1 was found in various tissues including roots, stems, leaves, and especially flowers (Garcia-Ortiz et al., 2001). However, the expression profile of AtOGG1 in seeds is still unclear. To address this issue, the expression of AtOGG1 was analysed during seed development and germination using real-time PCR. In the developing siliques, the expression of AtOGG1 was detected from 5 to 20 days after pollination, with the highest transcript levels observed 15 days after pollination (Fig. 2). During seed germination, the expression of AtOGG1 was strongly increased compared to that of dry seeds (20 days after pollination), and high levels of expression for 24 h after imbibition (Fig. 2).
Real-time PCR analysis of AtOGG1 transcripts in developing siliques and imbibing seeds. Data represent three biological replicates each consisting of three technical replicates (n=9). Error bars represent the standard error of the means.
AtOGG1–YFP fusion protein was localized in the nucleus
The full-length cDNA of AtOGG1 was obtained by RT-PCR. A putative nucleus localization signal peptide (KRPRP) was identified in the N-terminus (Garcia-Ortiz et al., 2001). We re-evaluated the possible targeting signals using the PSORT program (version WoLF, http://psort.hgc.jp/), and signals for nucleus, mitochondria, and chloroplast were predicted. To investigate the intracellular localization of AtOGG1 in plant cells, YFP was fused to the C-terminus of AtOGG1 to construct an expression vector, AtOGG1–YFP, for transient expression in protoplasts prepared from Arabidopsis suspension-cultured cells. We co-expressed AtOGG1–YFP in Arabidopsis protoplasts with a fluorescent marker protein that would mark the nucleus [SV40-monomeric red fluorescent protein (mRFP); Kalderon et al., 1984]. The fluorescence of AtOGG1–YFP fusion protein colocalized with the nucleus marker SV40 (Fig. 3A), which demonstrated a clear localization of AtOGG1 in the nucleus. It has been reported that the three isoforms of hOGG1 were localized in the mitochondria, while only type 1a was found in the nucleus (Takao et al., 1998; Nishioka et al., 1999). In addition to the nuclear genome, plant cells possess mitochondria and chloroplast DNA which can be the targets of oxidative damage by ROS and should be repaired. To further document the localization of AtOGG1, we co-expressed the AtOGG1–YFP with fluorescent marker proteins for chloroplasts (RecA-mRFP; Kohler et al., 1997) and mitochondria (F1 ATPase-mRFP; Jin et al., 2003), or stained the mitochondria with Mitotracker. Essentially, our results showed no fluorescent signal in the chloroplasts (Fig. 3B) or mitochondria (Fig. 3C, 3D). Moreover, the YFP fluorescence displayed a distinctly punctate pattern within the nucleus. A similar result was observed with the localization of several chloroplast DNA glycosylase/AP lyases (Gutman and Niyogi, 2009). It has been observed that hOGG1 protein rapidly accumulates at sites of laser-induced oxidative DNA damage (Zielinska et al., 2011). The punctate pattern of AtOGG1 fluorescence implies that AtOGG1 may be associated with oxidized genomic DNA.
Subcellular localization of AtOGG1–YFP in Arabidopsis protoplasts. (A–C) Arabidopsis protoplasts were co-electroporated with AtOGG1–YFP and the DNA of a single organelle marker as indicated. (D) Arabidopsis protoplasts transfected with AtOGG1–YFP were stained with Mitotracker which was used to indicate the location of mitochondria. After 13–16 h of expression, the protoplasts were observed by confocal laser scanning microscope. Chlo, chloroplast; DIC, differential interference contrast; Mito, mitochondria. Scale bar 50 μm.
Isolation and characterization of transgenic plants overexpressing AtOGG1
To further investigate the in vivo functions of AtOGG1 in seeds, we generated transgenic Arabidopsis plants overexpressing AtOGG1 under the control of the constitutive cauliflower mosaic virus 35S promoter. Three independent transgenic lines (OE-1, OE-2, and OE-3) showing high expression levels of AtOGG1 in seeds were selected for further analysis. Real-time PCR analysis demonstrated that the transcript levels of AtOGG1 in transgenic seeds were 10-fold greater than those of wild-type seeds (Fig. 4A). However, the phenotype of transgenic plants was indistinguishable from wild-type plants under normal growth conditions (data not shown).
Genes expression and quantification of 8-oxo-dG in dry mature seeds of wild-type and transgenic plants overexpressing AtOGG1. (A) Real-time PCR analysis of AtOGG1 and AtMMH expression in dry mature seeds of transgenic and wild-type (wt) plants. The expression levels of AtOGG1 and AtMMH in wild-type plants were normalized to 1.0. Values represent three biological replicates each consisting of three technical replicates (mean±SD, n=9). (B) Quantification of 8-oxo-dG in dry mature seeds of transgenic and wild-type plants. Values are from three biological replicates each using 5 μg of DNA extracted from 80 mg of seeds (mean±SD). Statistical significance of differences was determined using Student’s t test and * indicates that the change in deviance was significant (*P < 0.05).
In the oxidized DNA induced by ROS, the 8-oxo-G combines with a deoxyribose in the deoxyguanosine, gives rise to 8-oxo-dG (Fig. 1A). 8-oxo-dG is a frequently used biomarker of oxidative DNA damage and can be detected by immunological techniques using the specific anti-8-oxo-dG antibody (Yoshida et al., 2002). Based on the chemical characteristics of 8-oxo-dG and the biological role of AtOGG1 (Dany and Tissier, 2001; Garcia-Ortiz et al., 2001) it is tempting to speculate that high AtOGG1 expression might be correlated with a low content of 8-oxo-dG. Using the quantification assay of 8-oxo-dG, we found that the levels of 8-oxo-dG in the dry mature seeds from transgenic Arabidopsis overexpressing AtOGG1 was significantly lower than those of wild-type seeds (Fig. 4B). In Arabidopsis another DNA glycosylase/AP lyase, AtMMH, which is a structural homologue of yeast MutM, has been reported (Ohtsubo et al., 1998). To further understand the correlation between the levels of 8-oxo-dG and the expression of AtOGG1, the transcript levels of AtMMH were analysed in dry mature seeds of transgenic lines and wild-type seeds. As shown in Fig. 4A, the expression levels of AtMMH were similar in both wild-type and transgenic seeds, suggesting that the lower content of 8-oxo-dG in the transgenic seeds was due to the increased AtOGG1 expression and not AtMMH.
Transgenic seeds overexpressing AtOGG1 exhibited enhanced seed longevity associated with reduced DNA damage
The in vivo functions of AtOGG1 in seeds were evaluated using CDT, which has been widely used for estimating seed storage potential or seed longevity (Bentsink et al., 2000; Tesnier et al., 2002; Oge et al., 2008; Rajjou and Debeaujon, 2008). Storage temperature and seed moisture content are key factors that control seed deterioration and viability loss during storage (Bradford et al., 1993; McDonald, 1999). Dry mature seeds of various lines were submitted to the storage treatment at 40 °C with 82% RH. All untreated seeds displayed nearly 100% germination in 2 days at 22 °C (Fig. 5A), indicating a high viability of the untreated seeds. After CDT for 5 days the germination rates (measured as a percentage of germination) of OE-1, OE-2, and OE-3 ranged between 60 and 75% compared with only 27% from those of the wild type at 7 days when germination of various lines reached the maximum level (Fig. 5B). The viability of seeds that failed to germinate were further estimated by tetrazolium assay and no living seeds was found (see Supplementary Fig. S1).
Characterization of AtOGG1-overexpressing lines. Dry mature seeds from transgenic lines overexpressing AtOGG1 and wild-type (wt) lines were submitted to the controlled deterioration treatment conducted at 15–20% moisture content (MC; 40 °C at 82% RH) for different numbers of days. (A) Germination percentages of seeds without CDT. (B) Germination percentages of seeds submitted to CDT for 5 days. The germination percentages were scored daily until they plateaued on the seventh day. (C) Germination percentages of seeds submitted to CDT for various durations. The germination percentages of these seeds were monitored 7 days after sowing. (D) Percentages of cotyledon expansion of surviving seeds submitted to CDT for 5 days. The data represent the ratio of the seeds with expanded cotyledons to the germinated seeds 10 days after sowing. (E) Phenotypes of 10 day-old seedlings grown on a 9 mm Petri dish containing half-strength MS media from seeds after 5 days of CDT. (F) Quantification of 8-oxo-dG in dry and 24 h-imbibed seeds of transgenic and wild-type plants. Values are from three biological replicates each using 5 μg of DNA extracted from 200 mg of seeds (mean±SD). For (A) and (B) the 0 day time point indicates the beginning of the experiment after seeds were surface-sterilized and stored for 2 days at 4 °C. The symbols used in (B) and (C) to depict the transgenic and wild-type lines are the same as in (A). For (A–D) values are from four technical replicates of 100 seeds each (4 × 100; mean±SD). For (D) and (F) statistical significance of differences was determined using Student’s t test and * indicates that the change in deviance was significant (*P <0.05, **P <0.01).
To further document this finding, CDT was performed for between 1 and 7 days on various lines and percentage germination was monitored 7 days after sowing. In response to varying lengths of CDT both transgenic and wild-type seeds displayed decreased rates of germination after 2 days (Fig. 5C), indicating a loss of seed viability. Similar results have been reported with dry mature seeds from various Arabidopsis seed databases (Tesnier et al., 2002; Oge et al., 2008; Rajjou et al., 2008; Sattler et al., 2004; Zhou et al., 2012). In the wild-type seeds CDT for 4–7 days resulted in a dramatic loss of germination potential, whereas only moderately decreased of germination potential was observed in transgenic lines (Fig. 5C). Aged seeds that germinate often suffer from loss of vigour (Berjak and Villiers, 1972; Chu et al., 2011; Zhou et al., 2012). Among the seeds that germinated after CDT, the percentages of seedlings with cotyledon expansion from the transgenic lines were much higher than those of the wild type (Fig. 5D, 5E), implying a better repair and recovery mechanism in the transgenic seeds. These results demonstrated that the transgenic seeds overexpressing AtOGG1 were more resistant to CDT than the wild-type seeds.
It has been suggested that ROS accumulation in aged seeds leads to the deleterious effects of oxidative stress (McDonald, 1999; Bailly, 2004; Lee et al., 2010). The content of 8-oxo-dG induced by CDT was assessed in dry and imbibing aged seeds of both wild-type and transgenic lines. After CDT treatment for 5 days, dry mature seeds of the wild type showed a substantially higher accumulation of 8-oxo-dG (7 pmol μg−1 DNA) than the transgenic seeds (2.5–4 pmol μg−1 DNA) (Fig. 5F). After 24 h of imbibition there was an increased level of 8-oxo-dG in both wild-type and transgenic seeds, suggesting possible oxidative activities during seed germination (Fig. 5F). However, the transgenic lines displayed only a slight increase in 8-oxo-dG levels (3–6 pmol μg−1 DNA) compared to wild-type seeds (16 pmol μg−1 DNA) (Fig. 5F).
Overexpression of AtOGG1 enhanced seed tolerance to abiotic stresses
The up-regulated expression of AtOGG1 during imbibition (Fig. 2A) indicated a role for AtOGG1 in the germination process. To investigate the role of AtOGG1 during germination, dry mature seeds were subjected to abiotic stress treatments. In the chemically induced stress treatments, MV was used to mimic oxidative stress due to the accumulation of superoxide anions and hydrogen peroxide (Espelund et al., 1995; Haslekas et al., 2003). NaCl and Man were used to mimic osmotic stresses that lead to the accumulation of ROS and ultimately affect seed germination (Price and Hendry, 1991; Borsani et al., 2001). In the presence of 100 μM MV, 175 mM NaCl, or 500 mM Man, germination was complete after 8 days for all genotypes (Fig. 6A, 6C, 6E). With 5 μM MV, 75 mM NaCl, or 150 mM Man, the percentages of germination in the wild-type and transgenic lines were similar (Fig. 6B, 6D, 6F). However, seeds overexpressing AtOGG1 were substantially more tolerant than the wild-type seeds to the increased concentrations of MV, NaCl, or Man (Fig. 6B, 6D, 6F). In response to 100μM MV the transgenic seeds displayed a significantly higher germination percentage in comparison with wild-type seeds (Fig. 6A, 6B). After 8 days 56–65% of the transgenic seeds germinated, whereas only about 31% of wild-type seeds germinated. Similar results were also observed in the presence of different concentrations of NaCl and Man (Fig. 6C, 6D, 6E, 6F).
Transgenic seeds overexpressing AtOGG1 displayed enhanced tolerance to abiotic stresses. Dry mature seeds from three independent AtOGG1-overexpressing transgenic lines and wild-type (wt) lines were germinated in the presence of an imposed stressor as indicated. Germination percentages were monitored daily after sowing until they did not increase, which was the eighth day. (A, C, E) Germination percentages of seeds sown on half-strength MS media supplied with 100 μM MV, 175 mM NaCl, or 500 mM Man, respectively. (B, D, F) Germination percentages of seeds sown on half-strength MS media supplied with different concentrations of MV, NaCl, or Man, respectively. (G) Germination percentages of seeds subjected to heat-stress treatments for 30 min. (H) Percentages of cotyledon expansion of surviving seeds subjected to heat-stress treatments for 30 min. The data represent the ratio of seeds with expanded cotyledons to germinated seeds 10 days after sowing (*P < 0.05, **P < 0.01; Student’s t test). For (B, D, F, G) the germination percentages were monitored 8 days after sowing. For (A–G) the symbols used for every line are indicated in (A) and values are from four technical replicates, each with 100 seeds (4× 100) (mean±SD).
Furthermore, an extreme thermotolerance assay was conducted to test the thermotolerance of transgenic seeds. Dry mature seeds from transgenic and wild-type lines were immerged into hot water with different temperatures for 30 min. Although the heat stress treatments slowed down the subsequent germination of all genotypes, seeds from transgenic lines were much more tolerant to heat stress than controls (Fig. 6G). In addition, the cotyledon expansion rates from the germinating transgenic seeds were significantly higher than those of wild-type seeds, and transgenic seedlings were also more vigorous than the wild type (Fig. 6H). Taken together, these data demonstrated that overexpression of AtOGG1 in Arabidopsis positively enhanced seed-germination ability under adverse conditions.
Evidence of up-regulation of genes involved in the BER and down-regulation of genes response to DNA damage in germinating seeds of transgenic lines
Previous studies in human and bacteria have revealed that there are several enzymes involved in the course of BER of DNA damaged by 8-oxo-G. After the excision of 8-oxo-G by DNA glycosylase, both AP endonuclease and DNA ligase are required for the completion of BER (Fig. 1B) (Lu et al., 2001). In Arabidopsis, an apurinic endonuclease-redox protein (AtARP) appears to constitute the major AP endonuclease in cell extracts (Cordoba-Canero et al., 2011). Three DNA ligase genes have been identified in Arabidopsis, termed AtLIG1, AtLIG4, and AtLIG6 (Taylor et al., 1998; West et al., 2000; Waterworth et al., 2010). However, only AtLIG1 has been proved to be required for the completion of BER (Cordoba-Canero et al., 2011). Using real-time PCR, we investigated the expression levels of both AtARP and AtLIG1 in imbibing seeds. Both AtARP and AtLIG1 transcripts showed no significant up-regulation in the dry mature seeds (Fig. 7A). However, after 6 h of imbibition the induction of AtLIG1 and AtARP transcripts in the three transgenic seeds was much higher than those of the wild-type seeds (Fig. 7B). It is tempting to speculate that the higher induction of transcripts of both genes, AtLIG1 and AtARP, may be required to complete the BER pathway initiated by the AtOGG1 in the overexpressing seeds.
Analysis of transcript levels of genes involving in the BER and response to DNA damage. (A) Real-time PCR analysis of transcript levels of genes as indicated in imbibing wild-type (wt) seeds. (B) Real-time PCR analysis of the induction of transcript levels of genes as indicated after 6 h of imbibition in wild-type and transgenic seeds, compared with those of dry mature seeds. Genes are indicated by the same symbols in both panels. Data represent three biological replicates and three technical replicates (n=9). Error bars represent the standard error of the means.
Although we have demonstrated that 8-oxo-dG accumulated in germinating seeds after CDT (Fig. 5), it is of interest to examine whether DNA damage can also occur in the imbibing seeds without CDT. To address this purpose, the expression of two DNA damage-response genes, AtRAD51 and AtPARP2, was examined. RAD51 functions as a factor participating in the homologous recombination repair of DNA double-strand breaks and PARP2 is a DNA signalling factor involving in the DNA repair process (Babiychuk et al., 1998; Bray and West, 2005). Both AtRAD51 and AtPARP2 have been found to be up-regulated in response to DNA damage and have been used as markers to indicate the occurrence of DNA damage (Waterworth et al., 2010, 2011). In the early germination stage of untreated wild-type seeds, both genes exhibited increased transcript levels (Fig. 7A), suggesting the possibility of DNA damage during seed germination. In contrast, the induction of AtRAD51 and AtPARP2 transcripts in the seeds from three transgenic plants imbibed for 6 h were significantly lower than those of the wild-type seeds (Fig. 7B). This suggested that overexpression of AtOGG1 in transgenic seeds resulted in reduced accumulation of DNA damage in imbibing seeds.
Discussion
Under normal conditions, oxidized DNA generated by ROS is believed to be the major source of DNA damage during seed storage and germination (Dandoy et al., 1987; Bray and West, 2005). 8-oxo-G, a predominant DNA lesion induced by ROS, mediates mispairing during DNA replication, impairing the genetic resource and necessitating repair by DNA repair enzymes. To combat the adverse effects of ROS on DNA, plants have evolved multiple DNA repair pathways to counteract continuous genome damage (Britt, 1996; Wood, 1996; Lee et al., 2010; Waterworth et al., 2011). The BER pathway is an essential cellular defence mechanism against oxidative DNA damage, especially 8-oxo-G-induced mutagenesis (Lu et al., 2001). AtOGG1 has been identified and enzymatically characterized as a DNA glycosylase/AP lyase involved in the BER pathway (Dany and Tissier, 2001; Garcia-Ortiz et al., 2001). Analysis of the expression pattern of AtOGG1 has disclosed increased transcript levels at the later development of seeds (15 days after pollination) and during early germination (Fig. 2A). Since seed dessication and imbibition are correlated with high levels of ROS (Waterworth et al., 2011) the increased expression of AtOGG1 during those processes suggests a role for AtOGG1 in repairing oxidative DNA damage in seeds.
Seed longevity is vital for germplasm conservation and is also an important trait for both ecological and agronomic aspects of crop growth (Oge et al., 2008; Rajjou et al., 2008; Waterworth et al., 2010; Kochanek et al., 2011). However, seeds gradually lose viability during storage (Berjak and Villiers, 1972; Waterworth et al., 2011), and this is greatly accelerated when seeds are subjected to harsh storage conditions (Tesnier et al., 2002; Sattler et al., 2004; Oge et al., 2008). Considering that DNA damage is associated with loss of seed viability during storage (Berjak and Villiers, 1972; Cheah and Osborne, 1978; Waterworth et al., 2011), we investigated the correlation between AtOGG1 and seed longevity. The CDT has been developed under laboratory conditions to mimic poor storage conditions of seeds by exposing seeds to high temperatures and high RH. It has been recognized as a good test used to predict the quality and longevity of seedlots. In the present study the CDT assay showed that aged transgenic seeds maintained significantly higher germination percentages than wild-type seeds (Fig. 5B, 5C). In addition, after 5 days of CDT a higher percentage of cotyledon expansion and more vigorous seedlings were observed in transgenic lines in comparison with the wild type (Fig. 5D, 5E). These data demonstrate that overexpression of AtOGG1 confers the transgenic lines with enhanced seed longevity compared to the wild-type lines. Similarly, several studies revealed that seed aging is associated with increased DNA damage in different species (Vijay et al., 2009; El-Maarouf-Bouteau et al., 2011). Recent study by Waterworth et al. (2010) has further confirmed that DNA ligase-mediated DNA repair is a determinant of seed longevity. Enhanced seed longevity through repair of the damage induced by ROS by overexpression of specific enzymes has been reported previously (Prieto-Dapena et al., 2006; Oge et al., 2008; Lee et al., 2010).
Previous studies have demonstrated the function of AtOGG1 in eliminating 8-oxo-G from DNA in vitro (Dany and Tissier, 2001; Garcia-Ortiz et al., 2001). Here, quantification of 8-oxo-dG revealed that overexpression of AtOGG1 resulted in reduced oxidative DNA damage in dry and imbibing transgenic seeds (Fig. 4B, 5F). The lower accumulation of 8-oxo-dG in dry mature seeds of transgenic plants is thought to be associated with the overexpression of AtOGG1 (Fig. 4B), indicating a potential in vivo role for AtOGG1 in repairing DNA damage mediated by 8-oxo-G. It has been reported that overexpressing AtOGG1 in a mutM mutY mutant could suppress its spontaneous mutator phenotype (Dany and Tissier, 2001; Garcia-Ortiz et al., 2001). Overexpression hOGG1 in human lung cancer cell could reduce the GC→TA transversions induced by 8-oxo-G (Yamane et al., 2003). The repair of oxidative DNA damage by OGG1 has also been directly visualized in the nuclei of live cells (Zielinska et al., 2011). The high level of AtOGG1 in the transgenic plants seemed to initiate an increased level of BER, which was indicated by the increased induction of transcript levels of AtARP and AtLIG1 (Fig. 7B), and thereby decreased the accumulation of 8-oxo-dG in dry mature seeds of transgenic lines. We also showed that the induced transcripts of DNA damage-response genes, AtRAD51 and AtPARP2, significantly decreased after imbibition of the transgenic seeds (Fig. 7B), indicating an improved repair ability in transgenic seeds overexpressing AtOGG1. A similar result has also been reported in a study on DNA ligase, which revealed that DNA damage-response genes were induced by the loss of ligase-mediated DNA repair in atlig6-1 and atlig4-5 atlig6-1 mutants (Waterworth et al., 2010). The enhanced seed longevity of transgenic lines may be due to the overexpressed AtOGG1-mediated repair by lowering the accumulation of DNA damage in the dry matured seeds during seed storage and enhanced ability to repair DNA lesions during seed germination.
As sessile organisms, plants are inevitably exposed to environmental stresses. A common effect of many environmental stresses is to cause oxidative damage. The presently observed phenotype of varying tolerance towards MV, NaCl, Man, and high temperature (Fig. 6) in germinating Arabidopsis seeds of three transgenic lines revealed a correlation between the accumulation level of AtOGG1 in dry mature seeds and seed germination vigor. During seed germination, repair of accumulated DNA damage is initiated at the earliest stages of imbibition (Osborne et al., 1984; Waterworth et al., 2011) and prior to the initiation of cell division in order to minimize impairment of subsequent seedling development (Waterworth et al., 2010). The reduced induction of 8-oxo-dG in the imbibing seeds of transgenic lines supports the hypothesis that the AtOGG1 present in the dry seeds could be functional as soon as the imbibed seeds become metabolically active during early germination (Fig. 4A, 5F). A study of DNA ligases has also demonstrated that the accumulation and repair of DNA damage influenced seed germination, and that both genome integrity and the ability to repair damaged DNA affected seed germination (Waterworth et al., 2010).
To further investigate the role of AtOGG1 in seeds, Arabidopsis plants with reduced levels of AtOGG1 transcripts and proteins were generated using an RNAi approach (see Supplementary Fig. S2). Although the AtOGG1-overexpressing lines displayed enhanced seed viability, AtOGG1-silenced lines showed no difference from the wild type with respect to seed longevity or tolerance to adverse condition (Supplementary Fig. 2D, 2E). The reduction of AtOGG1 protein did not influence the level of 8-oxo-dG in the silenced lines (Supplementary Fig. 2C). Consistent with our observations, Murphy (2005) reported that an AtOGG1-knockout mutant (termed Ogg2) displayed no phenotypic difference from the wild type in growth, development, or reproductive potential under adverse conditions. Furthermore, the double mutant, which lacked both DNA glycosylase/AP lyases, AtOGG1 and AtMMH, also failed to show a difference from the wild type (Murphy, 2005). The similar results were observed in other organisms (Klungland et al., 1999; Friedberg and Meira, 2003). The knockout mice defective in OGG1 showed no indication of pathology but a slower repair of 8-oxo-G (Klungland et al., 1999). In yeast, a yeast strain defective in ntg1 ntg2 apn1, although exhibiting a mutant phenotype, did not show any sensitivity to the oxidizing agents H2O2 and menadione (Swanson et al., 1999). In Arabidopsis, although AtOGG1 and AtMMH displayed substrate specificity, they are functionally redundant (Murphy and George, 2005). In addition, 16 homologues of DNA glycosylase genes have been found in the Arabidopsis genome (Murphy, 2005). Recently, research into Arabidopsis has reported that both single and triple mutants of three chloroplast DNA glycosylases (AtNTH1, AtNTH2, and AtARP) failed to exhibit a different phenotype from wild-type plants (Gutman and Niyogi, 2009). Seemingly unaffected DNA that has previously been subjected to oxidative stress may be ascribed to the redundant nature of the BER pathway in Arabidopsis. This speculation may explain why there is no observed difference in the expression levels of genes involving BER and DNA damage response between the silenced lines and the wild type (Supplementary Fig. 2F). Furthermore, it is likely that there are other DNA repair pathways, such as nucleotide excision repair and recombinational repair, would complement the lack of AtOGG1 (Lu et al., 2001).
Altogether, these data strongly suggest that overexpression of AtOGG1 in Arabidopsis enhanced not only seed longevity but also seed tolerance to adverse conditions. Moreover, consistent with previous studies, our work demonstates a close link between seed longevity during dry storage and tolerance to abiotic stress during germination (McDonald, 1999; Clerkx et al., 2004; Oge et al., 2008; Rajjou et al., 2008).
Supplementary material
Supplementary material is available at JXB online.
Supplementary Fig. S1. Viability test of the seed samples that failed to germinate.
Supplementary Fig. S2. Characterization of AtOGG1-silenced lines.
Abbreviations
- AP
apurinic/apyrimidinic
- AtOGG1
Arabidopsis thaliana OGG1
- BER
base excision repair
- CDT
controlled deterioration treatment
- hOGG1
human OGG1
- 8-oxo-dG
8-hydroxy-2′-deoxyguanosine
- 8-oxo-G
7,8-dihydro-8-oxoguanine
- Man
mannitol
- mRFP
monomeric red fluorescent protein
- MS medium
Murashige and Skoog medium
- MV
methyl viologen
- ORF
open reading frame
- RH
relative humidity
- RNAi
RNA interference
- ROS
reactive oxygen species
- YFP
yellow fluorescent protein
- yOGG1
yeast OGG1
This work was supported by grants from Guangdong Agriculture Science and Technology Team Project (2011A02010210), the Natural Science Foundation of Guangdong Province (9151027501000075), the Guangdong Provincial Science and Technology Program (2010D020301003), and the National Natural Science Foundation of China (30370912).
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