DNA Repair 21 (2014) 171–176
Contents lists available at ScienceDirect
DNA Repair journal homepage: www.elsevier.com/locate/dnarepair
Brief Communication
Plant DNA-damage repair/toleration 100 protein repairs UV-B-induced DNA damage Nozomi Fujimori 1 , Nana Suzuki 1 , Yuko Nakajima, Shunji Suzuki ∗ Laboratory of Fruit Genetics Engineering, The Institute of Enology and Viticulture, University of Yamanashi, Kofu, Yamanashi 400-0005, Japan
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Article history: Received 26 November 2013 Received in revised form 11 April 2014 Accepted 28 May 2014 Available online 17 June 2014 Keywords: Abasic sites DNA damage DNA-damage repair/toleration 100 DNA single-strand breaks Grape Leucine-rich repeat UV-B
a b s t r a c t We report the characterization of VvDRT100-L, a grape DNA-damage repair/toleration 100 protein. VvDRT100-L has nine leucine-rich repeats and belongs to the plant DRT100 protein family. VvDRT100-L is expressed abundantly in green organs of grapevines, including tendrils, leaves, and green berry skins. The overexpression of VvDRT100-L in Arabidopsis plants decreased the number of abasic sites and the frequency of DNA single-strand breaks in the DNA damaged by UV-B irradiation, whereas UV-B irradiation markedly increased the number of abasic sites and the frequency of DNA single-strand breaks in T-DNA insertion mutant drt100 plants. VvDRT100-L-overexpressing plants remained viable and noticeably healthy under lethal UV doses, suggesting that VvDRT100-L may enhance UV tolerance in plant. Taken together, we concluded that VvDRT100-L might play an important role in the repair and toleration of UV-B-induced DNA damage. These findings would help us better understand how plants acquire UV stress acclimation, tolerance and DNA repair. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Phenolic compounds in grape berry skins are important secondary metabolites for wine quality. The compounds affect the taste and mouth-feel properties of wines as well as the stability of color and quality of stored wines [1]. Phenolic compounds also play an important role as UV-screening agents to protect plants from UV damage [2]. UV damage includes DNA damage, the suppression of photosynthesis, the generation of reactive oxygen species, and abnormal photomorphogenesis [3]. In grapevine, the accumulation of UV-absorbing phenolic compounds, particularly hydroxycinnamic acids, in berry skins resulted in efficient shielding against UV-A [2]. However, the shielding of grape berries against UV-B was incomplete because UV-B irradiation downregulated the synthesis of hydroxycinnamic acids in berry skins [2]. Thus, at least for UV-B irradiation, phenolic compounds appear to not protect grape berries from damage induced by UV-B irradiation.
Abbreviations: DRT100, DNA-damage repair/toleration 100; LRR, leucine-rich repeat; UV-B, ultraviolet-B. ∗ Corresponding author. Tel.: +81 55 220 8394; fax: +81 55 220 8768. E-mail address: [email protected] (S. Suzuki). 1 These authors contributed equally to the article. http://dx.doi.org/10.1016/j.dnarep.2014.05.009 1568-7864/© 2014 Elsevier B.V. All rights reserved.
DNA damage by UV irradiation causes deleterious mutation in plants. To avoid the severity of DNA damage induced by UV irradiation, plants are equipped with various DNA damage repair systems. Homologous recombination mediated by RecA-mediated DNA repair machinery is the most well characterized DNA damage repair system [4]. Plant RecA proteins form a small family. In Arabidopsis, five putative RecA homologues were identified in the genome and predicted to be localized in mitochondria and chloroplasts [5,6]. DNA-damage repair/tolerance 100 (DRT100) is one of the plant RecA proteins and localizes in chloroplasts [5]. A complementation experiment of Arabidopsis DRT100 in a UV-sensitive recA mutant of Escherichia coli revealed that DRT100 partially restored UV resistance of the recA mutant [5,7]. However, to date, the fundamental functions of plant DRT100 in plant itself are yet to be elucidated. We have identified abiotic-stress-induced proteins containing leucine-rich repeats (LRRs) in grapevines and characterized the functions of the proteins in grapevines exposed to abiotic stress. By chance, we found that one deduced amino acid sequence of the proteins was similar to that of plant DRT100 protein. Our aim in the present study was to characterize the physiological role of grape DRT100 (named VvDRT100-L) under UV stress. Here, we demonstrated that VvDRT100-L played an important role in the DNA damage repair and toleration systems of the plant cells exposed to UV-B.
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2. Materials and methods 2.1. Plant materials Grapevines of Vitis vinifera cv. Merlot were cultivated in the experimental vineyard of The Institute of Enology and Viticulture, University of Yamanashi, Japan. Plant organs, including tendrils, young leaves, and stems, were collected from the grapevines on June 27, 2011. Green-yellow and black berries were collected from a single cluster on August 11, 2011. Berry skins were peeled off with a razor. Seeds were isolated from the black berries. Root samples were obtained from own-root cuttings of Merlot. 2.2. UV irradiation Plant materials were exposed to UV-B (280–320 nm, Panasonic, Osaka, Japan) for 15 min at room temperature. The intensity of UVB radiation was 200–230 W/cm2 . As control experiment, plant materials were incubated in the dark for 15 min at room temperature. After UV irradiation, the plant materials were incubated under the indicated condition. 2.3. RNA isolation Plant materials were placed in a mortar containing liquid nitrogen and homogenized with a pestle. Approximately 100 mg of the pulverized tissue was transferred to a microtube. Total RNA was isolated using a NucleoSpin RNA Plant kit (Takara, Otsu, Japan) according to the manufacturer’s instructions. 2.4. Real-time RT-PCR Real-time RT-PCR analysis was performed with the procedure described previously [8]. Nucleotide sequences of the primers used in this study were as follows: V. vinifera DRT100-like protein (VvDRT100-L) primers (5 -CTCGCCGATTTTGACCTCTC-3 and 5 -TGTTGAGTCCGGTGTTGCTC-3 , GenBank accession no. JQ326308, respectively), and V. vinifera -actin primers (5 -CAAGAGCTGGAAACTGCAAAGA-3 and 5 -AATGAGAGATGGCTGGAAGAGG-3 , GenBank accession no. AF369524, respectively). -Actin was used for normalization and the expression level of each gene was expressed as a relative value. 2.5. Cluster analysis Cluster analysis of VvDRT100-L with plant DRT100 proteins and other DRT family proteins was performed. Briefly, the amino acid sequences of these proteins were collected from an NCBI database. The sequences were subjected to the neighbor-joining (NJ) method together with the predicted amino acid sequence of VvDRT100-L using Molecular Evolutionary Genetics Analysis software, MEGA4 [9]. 2.6. Overexpression of VvDRT100-L in Arabidopsis thaliana The open reading frame of VvDRT100-L was amplified from total RNA isolated from the UV-irradiated leaves by RT-PCR. The nucleotide sequences of the primers used were as follows: 5 -CATATGAAACTCTTCTTGTTCC-3 containing an NdeI site (underlined) and 5 -GTCGACTTACCAGAGACAGTCGTTGA-3 containing a SalI site (underlined). The PCR product was ligated into the pGEMT Easy vector (Promega, Madison, WI) and sequenced. Then, the PCR product was digested with NdeI and SalI and ligated into the NdeI and SalI sites of the binary vector pRI101-AN (Takara), resulting in a plant expression plasmid. The plant expression plasmid was transformed into Agrobacterium tumefaciens strain LBA4404.
A. thaliana Col-0 was transformed with the Agrobacterium by the floral dip method [10]. Three independent T3 homozygous lines were obtained and used for the DNA damage experiment and the UV protection assay. One transgenic line transformed with pRI101AN and one T-DNA insertion mutant line, drt100 (SALK 021479C), were used as the control plants. 2.7. Determination of the number of abasic sites Based on the formation of abasic sites by exposure of UV to DNA [11], DNA damage by UV-B irradiation was quantified using a DNA damage quantification kit (Dojindo, Kumamoto, Japan). Briefly, Arabidopsis transformants were exposed to UV-B at room temperature for 15 min as described above. After UV-B irradiation, genomic DNA was immediately isolated from the plants using a NucleoSpin Plant II kit (Takara). One g of genomic DNA was subjected to the determination of the number of abasic sites in the DNA using the kit according to the manufacturer’s instructions. Aldehyde reactive probe, N -aminooxymethylcarbonylhydrazin-D-biotin was used as a probe for abasic sites. The probe binds to abasic sites in the DNA through an aldehyde group present on the open ring form of the abasic sites. Finally, the number of abasic sites per 100,000 bp in the DNA was calculated using the calibration curve of standard DNA provided in the kit as a supplement. 2.8. Assessment of DNA single-strand breaks DNA single breaks and their repair were assessed using the procedure of Schröder et al. [12]. Briefly, Arabidopsis transformants were exposed to UV-B at room temperature for 15 min as described above. Genomic DNA was immediately isolated from the plants. Thirty ng of genomic DNA was subjected to the determination of DNA single-breaks in the DNA according to the procedure of fast micromethod DNA single-strand break assay [12]. Finally, strand scission factor (SSF) values were calculated using the following formula: SSF = log
% of double-stranded DNA in the sample % of double-stranded DNA in the control
Unwounded DNA of wild type plants was used as control. SSF values were calculated after a denaturation period of 20 min. 2.9. Detection of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidine photoproducts Cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidine photoproducts (6-4PP) were detected using an OxiSelect UV-Induced DNA Damage ELISA Combo Kit (CPD/6-4PP Quantitation, Cell Biolabs, San Diego, CA). Briefly, Arabidopsis transformants were exposed to UV-B at room temperature for 15 min as described above and immediately subjected to DNA isolation. Two g of Genomic DNA isolated from the plants was subjected to the detection of CPD-DNA or 6-4PP-DNA using the kit according to the manufacturer’s instructions. Finally, the amount of CPD-DNA or 64PP-DNA in the DNA was calculated using the calibration curve of standard denatured CPD-DNA or 6-4PP-DNA provided in the kit as a supplement. 2.10. UV protection assay T4 seeds of the transformants were plated on MS medium in a six-well plate and incubated at 22 ◦ C for 3 d in an incubator (11.8 W m−2 /16 h/d). Then, the plate was exposed to UV-B (220 W/cm2 ) for 4 h at room temperature. After UV irradiation, the plate was returned to the incubator. Viability was assessed at 14 d after re-incubation.
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Fig. 1. Identification of VvDRT100-L. (A) Deduced amino acid sequence of VvDRT100-L. The LRR N-terminal domain (LRRNT) is boxed. The nine LRRs (LRR1–LRR9) are underlined. (B) Cluster analysis. By means of the NJ method using MEGA4, a phylogenetic tree was constructed from the deduced amino acid sequences. Populus deltoides DRT100 (accession no. ABS18952). Glycine max DRT100 (accession no. NP 001241499). Medicago truncatula DRT100 (accession no. XP 003629372). Arabidopsis thaliana DRT100 (accession no. NP 187867). A. lyrata subsp. lyrata DRT100 (accession no. XP 002884914). A. thaliana DRT111 (accession no. NP 174336). A. thaliana DRT112 (accession no. NP 173459). (C) Transcription profile of VvDRT100-L in grapevine organs. Total RNA was isolated from the indicated organs and subjected to real-time RT-PCR analysis. -Actin was used as internal control. Data were calculated as gene expression relative to ˇ-actin expression. Bars indicate means ± standard deviation of triplicate experiments. skin(G), green berry skins; skin(B), black berry skins.
2.11. Statistics Data in figures are expressed as means ± standard deviations. Statistical analysis was performed with the non-parametric Dunnett’s multiple comparison test using Excel statistics software (Social Survey Research Information, Tokyo, Japan). 3. Results 3.1. Identification of gene encoding DRT100 protein in grapevine We obtained the full length of the cDNA fragment containing a coding sequence (1071 bp) from grapevine, similar to plant DRT100 protein. The deduced amino acid sequence had an LRR N-terminal domain and nine LRRs in the sequence (Fig. 1A). Cluster analysis of the deduced amino acid sequence with those of plant DRT proteins demonstrated that the sequence formed a cluster with plant DRT100 proteins, not with DRT111 and DRT112 proteins (Fig. 1B). We found two nucleotide sequences (LOC100241571, GenBank accession no. XM 002285517 and LOC100855260, GenBank accession no. XM 003633501) that were predicted to be grape DRT100-like proteins by the grape genome project, in the NCBI
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Fig. 2. Overexpression of VvDRT100-L repairs abasic sites induced by UVB irradiation in Arabidopsis plants. (A) Transcription profile of VvDRT100-L in VvDRT100-L-overexpressing plants. Three independent Arabidopsis lines overexpressing VvDRT100-L (OE1, OE2, and OE3) were generated. Total RNA was isolated from 12-day-old seedlings non-treated by UV-B and subjected to real-time RTPCR analysis. -Actin was used as internal control. Data were calculated as gene expression relative to -actin expression. Bars indicate means ± standard deviation of triplicate experiments. (B) Number of abasic sites. Twelve-day-old seedlings were exposed to UV-B and the numbers of abasic sites in the DNA isolated from the seedlings were measured. Wild type Col-0 (wild), Arabidopsis line transformed with pRI101-AN vector (pRI), and T-DNA insertion mutant drt100 (SALK 021479C) were used as control plants. Bars indicate means ± standard deviation of triplicate experiments. *p < 0.05 as compared with non-treated plant.
nucleotide database. LOC100241571 and LOC100855260 appear on chromosomes 6 and 13, respectively. Alignment analysis of the three nucleotide sequences revealed that our cloned cDNA showed a perfect match to LOC100855260. Based on these results, the cDNA sequence identified in the present study was deposited in GenBank database as V. vinifera DRT100-like protein, VvDRT100-L (GenBank accession no. JQ326308). VvDRT100-L transcripts were detected in various organs of grapevine (Fig. 1C). Green organs, including tendrils, leaves, and young berry skins, except stems, abundantly expressed VvDRT100L. In contrast, VvDRT100-L was not abundantly expressed in seeds, roots, stems, and matured black berry skins. From the organ distribution of VvDRT100-L transcripts, it is plausible that VvDRT100-L exists abundantly in ‘green’ organs frequently exposed to sunlight. 3.2. VvDRT100-L overexpression repairs DNA damage caused by UV-B irradiation To determine whether VvDRT100-L has the ability to repair DNA damage induced by UV-B irradiation, three independent Arabidopsis lines overexpressing VvDRT100-L (OE1, OE2, and OE3) were generated. All transformants overexpressed VvDRT100-L transcripts, although the transcriptional levels were different among lines (Fig. 2A).
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We also detected CPD and 6-4PP in the DNA of UV-B UVirradiated overexpressing plants. UV-B irradiation increased the amount of CPD-DNA (Fig. 4A) and 6-4PP-DNA (Fig. 4B) in the DNA of wild plants, pRI, and three VvDRT100-L-overexpressing Arabidopsis plants. The amount of CPD-DNA and 6-4PP-DNA in the DNA of OE1 with UV-B irradiation decreased compared with those of wild and pRI control plants (Fig. 4, OE1 + UV-B). However, the amounts of CPD-DNA and 6-4PP-DNA in the DNA of OE2 and OE3 with UV-B irradiation increased compared with those of wild and pRI control plants (Fig. 4, OE2 + UV-B and OE3 + UV-B). Also, there was no difference of the amounts of CPD-DNA and 6-4PP-DNA in the DNA between OE1 and drt100 (Fig. 4, drt100). Taken together, these results suggested that VvDRT100-L might not repair CPD-DNA and 6-4PP-DNA induced by UV-B irradiation, Fig. 3. Overexpression of VvDRT100-L decreases DNA single-strand breaks induced by UV-B irradiation in Arabidopsis plants. Twelve-day-old seedlings were exposed to UV-B and the frequency of DNA single-strand breaks (SSF value) in the DNA isolated from the seedlings was determined. Unwounded DNA of wild type plants was used as control. Bars indicate means ± standard deviation of four independent experiments with two seedlings. *p < 0.05 as compared with pRI.
UV-B irradiation increased the number of abasic sites in the DNA of Arabidopsis wild plants and pRI101-AN vector transformed plants (Fig. 2B, wild and pRI). In all of the three VvDRT100-Loverexpressing Arabidopsis plants, the numbers of abasic sites in the DNA without UV-B irradiation were less than those of control plants, although the difference was minimal compared with control plants (Fig. 2B, OE1, OE2, and OE3). The numbers of abasic sites in the DNA of the overexpressing plants were decreased by UV-B irradiation (Fig. 2B, OE1 + UV-B, OE2 + UV-B, and OE3 + UV-B). In the T-DNA insertion mutant drt100, UV-B irradiation markedly increased the number of abasic sites in the DNA (Fig. 2, drt100). DNA single-strand breaks are also generated during excision repair of abasic sites from the DNA damaged by UV irradiation [13]. The frequencies of DNA single-strand breaks in all of the overexpressing plants decreased under UV-B irradiation compared with that of pRI (Fig. 3). In the DNA of drt100, the breaks by UV-B irradiation increased compared with that of pRI.
3.3. VvDRT100-L overexpression confers tolerance to lethal UV-B irradiation in plants To evaluate whether VvDRT100-L protects plant cells from UV damage induced by lethal UV-B irradiation, we performed UV protection assay using the three VvDRT100-L-overexpressing lines. Lethal UV-B irradiation resulted in withered hypocotyls and leaves of drt100 mutant (Fig. 5). The growth of wild plants and pRI101AN vector transformed plants were also suppressed by lethal UV-B irradiation. Two of VvDRT100-L overexpressing seedlings (OE2 and OE3), especially OE2, were noticeably healthy even after lethal UVB irradiation. However, OE1 show only a modest toleration against lethal UV irradiation. 4. Discussion Plants require sunlight for photosynthesis. As UV-B is predominantly absorbed by stratospheric ozone, a very small dose reaches plants on Earth. However, due to the rapid depletion of the ozone layer over the past two decades, a large dose of UV-B has reached the Earth, resulting in unavoidable damage to plants exposed to it [14]. To avoid the deleterious effects of UV-B on macromolecules in plant cells, plants produce a large number of UV-B-absorbing
Fig. 4. Overexpression of VvDRT100-L has no effect on the repair of (A) CPD-DNA and (B) 6-4PP-DNA induced by UV-B irradiation in Arabidopsis plants. Twelve-day-old seedlings were exposed to UV-B and the amounts of CPD-DNA or 6-4PP-DNA in the DNA isolated from the seedlings were measured. Wild type Col-0 (wild), Arabidopsis line transformed with pRI101-AN vector (pRI), and T-DNA insertion mutant drt100 were used as control plants. Bars indicate means ± standard deviation of four independent experiments.
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Fig. 5. Overexpression of VvDRT100-L confers tolerance to lethal UV-B irradiation in Arabidopsis plants. Three-day-old seedlings after plating were exposed to UV-B for 4 h at room temperature. Viability was assessed at 14 d after re-incubation. The results were reproducible in three independent experiments. Scale bars = 1 cm.
phenolic compounds. Arabidopsis mutant uvt1 acquired tolerance to lethal UV-B irradiation by increasing the number of UVabsorbing phenolic pigments [15]. UV-B irradiation also caused DNA damage in plant cells. In the present study, we demonstrated that UV-B irradiation of plants led to DNA damage through the formation of abasic sites (Fig. 2), the generation of DNA single-strand breaks (Fig. 3), and the production of CPD and 6-4PP (Fig. 4), in the DNA. Homologous recombination by the RecA-mediated DNA repair machinery is the most well characterized DNA damage repair system [4]. The present study is the first to show that plant DRT100, a member of plant RecA proteins, plays important roles in DNA damage repair and toleration against UV-B irradiation. Only a few reports related to plant DRT protein family have been published [4,5,7]. To our knowledge, Pang et al. [5] were the first to identify the Arabidopsis DRT100 gene; they showed that DRT100 partially complemented the growth phenotype of E. coli recA mutants under UV irradiation. The overexpression of VvDRT100-L in Arabidopsis plants enhanced the activity of the DNA damage repair system under UV-B irradiation (Figs. 2 and 3). Reciprocally, the drt100 mutant, in which DRT100 transcripts were reduced by 24-fold compared with the wild type [4], lost its DNA damage repair activity and showed an increase in the number of abasic sites and the frequency of DNA singlestrand breaks in the DNA under UV-B irradiation. Accordingly, we estimated that VvDRT100-L might play an important role in repairing DNA damage induced by UV-B irradiation. As the oxidative damage of DNA led to the formation of abasic sites in the DNA [10], UV-B irradiation might induce the production of reactive oxygen species in plant cells [16]. This oxidative attack on deoxyriboses released free bases from DNA, resulting in strand breakage and the generation of abasic sites [10]. Based on this biochemical reaction of DNA induced by UV-B irradiation, the overexpressed VvDRT100-L protein may function as one of the master proteins in homologous recombination, which is important for the repair of strand DNA breakage, similarly to RecA in E. coli [17]. The numbers of abasic sites in the DNA of VvDRT100-Loverexpressing plants were decreased by UV-B irradiation as compared with non-irradiated plants (Fig. 2). UV-B irradiation induced the formation of abasic sites (Fig. 2), the generation of
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DNA single-strand breaks (Fig. 3), and the production of CPD and 6-4PP (Fig. 4), in the DNA of Arabidopsis plant. In plants, direct DNA repair machinery, such as nucleotide excision repair and homologous recombination, is activated to maintain genome integrity under UV stress [18]. However, VvDRT100-L did not repair CPD-DNA and 6-4PP-DNA induced by UV-B irradiation. Therefore, VvDRT100-L might specifically play a role in the repair/reduction of abasic sites and DNA single-strand breaks in plants. Taken together, these results raises a hypothesis that VvDRT100 interacts with UVB-induced or activated DNA repair machinery, resulting in the decrease of abasic sites and the frequency of DNA single-strand breaks in the overexpressing plants. Future investigation of the function of VvDRT100-L in vitro or in vivo is required and may provide new information on the DNA damage repair system in plants exposed to UV-B. The overexpression of VvDRT100-L resulted in the acquisition of tolerance to lethal UV-B irradiation in Arabidopsis (Fig. 5, OE2 and OE3). In contrast to the repair of DNA damage induced by short term UV-B irradiation, a great number of VvDRT100-L might be required for the acquisition of tolerance to lethal UV-B irradiation. This incompatibility may suggest the multiple functions of plant DRT100 in UV damage repair/toleration machinery of plants. Since it has been not reported that plant DRT100 confers UV damage tolerance in plants, we need further experiment focusing on both the cell death pathway induced by lethal UV-B irradiation and the mechanism involved in the acquisition of UV-B damage tolerance in plants. Conflicts of interest statement The authors declare that they have no conflicts of interest. Acknowledgement We are grateful to Mr. Keisuke Sugiyama of the University of Yamanashi for helpful discussions. References [1] S. Vidal, L. Francis, S. Guyot, N. Marnet, M. Kwiatkowski, R. Gawel, V. Cheynier, E.J. Waters, The mouth-feel properties of grape and apple proanthocyanidins in wine-like medium, J. Sci. Food Agric. 83 (2003) 564–573. ´ M. Riederer, E.E. Pfündel, UV screening by phenolics in [2] C.A. Kolb, J. Kopecky, berries of grapevine (Vitis vinifera), Funct. Plant Biol. 30 (2003) 1177–1186. [3] H. Frohnmeyer, D. Staiger, Ultraviolet-B radiation-mediated responses in plants. Balancing damage and protection, Plant Physiol. 133 (2003) 1420– 1428. [4] B.A. Rowan, D.J. Oldenburg, A.J. Bendich, RecA maintains the integrity of chloroplast DNA molecules in Arabidopsis, J. Exp. Bot. 61 (2010) 2575–2588. [5] Q. Pang, J.B. Hays, I. Rajagopal, A plant cDNA that partially complements Escherichia coli recA mutations predicts a polypeptide not strongly homologous to RecA proteins, Proc. Natl. Acad. Sci. U.S.A. 89 (1992) 8073–8077. [6] V. Shedge, M. Arrieta-Montiel, A.C. Christensen, S.A. Mackenzie, Plant mitochondrial recombination surveillance requires unusual RecA and MutS homologs, Plant Cell 19 (2007) 1251–1264. [7] Q. Pang, J.B. Hays, I. Rajagopal, T.S. Schaefer, Selection of Arabidopsis cDNAs that partially correct phenotypes of Escherichia coli DNA-damage-sensitive mutants and analysis of two plant cDNAs that appear to express UV-specific dark repair activities, Plant Mol. Biol. 22 (1993) 411–426. [8] M. Kohno, H. Takato, H. Horiuchi, K. Fujita, S. Suzuki, Auxin-nonresponsive grape Aux/IAA19 is a positive regulator of plant growth, Mol. Biol. Rep. 39 (2012) 911–917. [9] K.J.D. Tamura, M. Nei, S. Kumar, MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0, Mol. Biol. Evol. 24 (2007) 1596–1599. [10] S.J. Clough, A.F. Bent, Floral dip: a simplified method for Agrobacteriummediated transformation of Arabidopsis thaliana, Plant J. 16 (1998) 735– 743. [11] H.A. Dahlmann, V.G. Vaidyanathan, S.J. Sturla, Investigating the biochemical impact of DNA damage with structure-based probes: abasic sites, photodimers, alkylation adducts, and oxidative lesions, Biochemistry 48 (2009) 9347– 9359. [12] H.C. Schröder, R. Batel, H. Schwertner, O. Boreiko, W.E.G. Müller, Fast micromethod DNA single-strand-break assay, Methods Mol. Biol. 314 (2006) 287–305.
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[13] E.C. Friedberg, G.C. Walker, W. Siede, DNA Repair and Mutagenesis, American Society for Microbiology Press, Washington, DC, 1995. [14] J.A. Pyle, Global ozone depletion: observation and theory, in: P.J. Lumbsden (Ed.), Plants and UV-B. Responses to Environmental Changes, Cambridge University Press, Cambridge, 1996, pp. 3–12. [15] K. Bieza, R. Lois, An Arabidopsis mutant tolerant to lethal ultraviolet-B levels shows constitutively elevated accumulation of flavonoids and other phenolics, Plant Physiol. 126 (2001) 1105–1115.
[16] B.R. Jordan, Review: Molecular response of plant cells to UV-B stress, Funct. Plant Biol. 29 (2002) 909–916. [17] M.M. Cox, Regulation of bacterial RecA protein function, Rev. Biochem. Mol. Biol. 42 (2007) 41–63. [18] J. Molinier, E. Lecher, E. Dumbliauskas, P. Genschik, Regulation and role of Arabidopsis CUL4-DDB1A-DDB2 in maintaining genome integrity upon UV stress, PLoS Genet. 4 (2008) e1000093.
Contents lists available at ScienceDirect
DNA Repair journal homepage: www.elsevier.com/locate/dnarepair
Brief Communication
Plant DNA-damage repair/toleration 100 protein repairs UV-B-induced DNA damage Nozomi Fujimori 1 , Nana Suzuki 1 , Yuko Nakajima, Shunji Suzuki ∗ Laboratory of Fruit Genetics Engineering, The Institute of Enology and Viticulture, University of Yamanashi, Kofu, Yamanashi 400-0005, Japan
a r t i c l e
i n f o
Article history: Received 26 November 2013 Received in revised form 11 April 2014 Accepted 28 May 2014 Available online 17 June 2014 Keywords: Abasic sites DNA damage DNA-damage repair/toleration 100 DNA single-strand breaks Grape Leucine-rich repeat UV-B
a b s t r a c t We report the characterization of VvDRT100-L, a grape DNA-damage repair/toleration 100 protein. VvDRT100-L has nine leucine-rich repeats and belongs to the plant DRT100 protein family. VvDRT100-L is expressed abundantly in green organs of grapevines, including tendrils, leaves, and green berry skins. The overexpression of VvDRT100-L in Arabidopsis plants decreased the number of abasic sites and the frequency of DNA single-strand breaks in the DNA damaged by UV-B irradiation, whereas UV-B irradiation markedly increased the number of abasic sites and the frequency of DNA single-strand breaks in T-DNA insertion mutant drt100 plants. VvDRT100-L-overexpressing plants remained viable and noticeably healthy under lethal UV doses, suggesting that VvDRT100-L may enhance UV tolerance in plant. Taken together, we concluded that VvDRT100-L might play an important role in the repair and toleration of UV-B-induced DNA damage. These findings would help us better understand how plants acquire UV stress acclimation, tolerance and DNA repair. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Phenolic compounds in grape berry skins are important secondary metabolites for wine quality. The compounds affect the taste and mouth-feel properties of wines as well as the stability of color and quality of stored wines [1]. Phenolic compounds also play an important role as UV-screening agents to protect plants from UV damage [2]. UV damage includes DNA damage, the suppression of photosynthesis, the generation of reactive oxygen species, and abnormal photomorphogenesis [3]. In grapevine, the accumulation of UV-absorbing phenolic compounds, particularly hydroxycinnamic acids, in berry skins resulted in efficient shielding against UV-A [2]. However, the shielding of grape berries against UV-B was incomplete because UV-B irradiation downregulated the synthesis of hydroxycinnamic acids in berry skins [2]. Thus, at least for UV-B irradiation, phenolic compounds appear to not protect grape berries from damage induced by UV-B irradiation.
Abbreviations: DRT100, DNA-damage repair/toleration 100; LRR, leucine-rich repeat; UV-B, ultraviolet-B. ∗ Corresponding author. Tel.: +81 55 220 8394; fax: +81 55 220 8768. E-mail address: [email protected] (S. Suzuki). 1 These authors contributed equally to the article. http://dx.doi.org/10.1016/j.dnarep.2014.05.009 1568-7864/© 2014 Elsevier B.V. All rights reserved.
DNA damage by UV irradiation causes deleterious mutation in plants. To avoid the severity of DNA damage induced by UV irradiation, plants are equipped with various DNA damage repair systems. Homologous recombination mediated by RecA-mediated DNA repair machinery is the most well characterized DNA damage repair system [4]. Plant RecA proteins form a small family. In Arabidopsis, five putative RecA homologues were identified in the genome and predicted to be localized in mitochondria and chloroplasts [5,6]. DNA-damage repair/tolerance 100 (DRT100) is one of the plant RecA proteins and localizes in chloroplasts [5]. A complementation experiment of Arabidopsis DRT100 in a UV-sensitive recA mutant of Escherichia coli revealed that DRT100 partially restored UV resistance of the recA mutant [5,7]. However, to date, the fundamental functions of plant DRT100 in plant itself are yet to be elucidated. We have identified abiotic-stress-induced proteins containing leucine-rich repeats (LRRs) in grapevines and characterized the functions of the proteins in grapevines exposed to abiotic stress. By chance, we found that one deduced amino acid sequence of the proteins was similar to that of plant DRT100 protein. Our aim in the present study was to characterize the physiological role of grape DRT100 (named VvDRT100-L) under UV stress. Here, we demonstrated that VvDRT100-L played an important role in the DNA damage repair and toleration systems of the plant cells exposed to UV-B.
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2. Materials and methods 2.1. Plant materials Grapevines of Vitis vinifera cv. Merlot were cultivated in the experimental vineyard of The Institute of Enology and Viticulture, University of Yamanashi, Japan. Plant organs, including tendrils, young leaves, and stems, were collected from the grapevines on June 27, 2011. Green-yellow and black berries were collected from a single cluster on August 11, 2011. Berry skins were peeled off with a razor. Seeds were isolated from the black berries. Root samples were obtained from own-root cuttings of Merlot. 2.2. UV irradiation Plant materials were exposed to UV-B (280–320 nm, Panasonic, Osaka, Japan) for 15 min at room temperature. The intensity of UVB radiation was 200–230 W/cm2 . As control experiment, plant materials were incubated in the dark for 15 min at room temperature. After UV irradiation, the plant materials were incubated under the indicated condition. 2.3. RNA isolation Plant materials were placed in a mortar containing liquid nitrogen and homogenized with a pestle. Approximately 100 mg of the pulverized tissue was transferred to a microtube. Total RNA was isolated using a NucleoSpin RNA Plant kit (Takara, Otsu, Japan) according to the manufacturer’s instructions. 2.4. Real-time RT-PCR Real-time RT-PCR analysis was performed with the procedure described previously [8]. Nucleotide sequences of the primers used in this study were as follows: V. vinifera DRT100-like protein (VvDRT100-L) primers (5 -CTCGCCGATTTTGACCTCTC-3 and 5 -TGTTGAGTCCGGTGTTGCTC-3 , GenBank accession no. JQ326308, respectively), and V. vinifera -actin primers (5 -CAAGAGCTGGAAACTGCAAAGA-3 and 5 -AATGAGAGATGGCTGGAAGAGG-3 , GenBank accession no. AF369524, respectively). -Actin was used for normalization and the expression level of each gene was expressed as a relative value. 2.5. Cluster analysis Cluster analysis of VvDRT100-L with plant DRT100 proteins and other DRT family proteins was performed. Briefly, the amino acid sequences of these proteins were collected from an NCBI database. The sequences were subjected to the neighbor-joining (NJ) method together with the predicted amino acid sequence of VvDRT100-L using Molecular Evolutionary Genetics Analysis software, MEGA4 [9]. 2.6. Overexpression of VvDRT100-L in Arabidopsis thaliana The open reading frame of VvDRT100-L was amplified from total RNA isolated from the UV-irradiated leaves by RT-PCR. The nucleotide sequences of the primers used were as follows: 5 -CATATGAAACTCTTCTTGTTCC-3 containing an NdeI site (underlined) and 5 -GTCGACTTACCAGAGACAGTCGTTGA-3 containing a SalI site (underlined). The PCR product was ligated into the pGEMT Easy vector (Promega, Madison, WI) and sequenced. Then, the PCR product was digested with NdeI and SalI and ligated into the NdeI and SalI sites of the binary vector pRI101-AN (Takara), resulting in a plant expression plasmid. The plant expression plasmid was transformed into Agrobacterium tumefaciens strain LBA4404.
A. thaliana Col-0 was transformed with the Agrobacterium by the floral dip method [10]. Three independent T3 homozygous lines were obtained and used for the DNA damage experiment and the UV protection assay. One transgenic line transformed with pRI101AN and one T-DNA insertion mutant line, drt100 (SALK 021479C), were used as the control plants. 2.7. Determination of the number of abasic sites Based on the formation of abasic sites by exposure of UV to DNA [11], DNA damage by UV-B irradiation was quantified using a DNA damage quantification kit (Dojindo, Kumamoto, Japan). Briefly, Arabidopsis transformants were exposed to UV-B at room temperature for 15 min as described above. After UV-B irradiation, genomic DNA was immediately isolated from the plants using a NucleoSpin Plant II kit (Takara). One g of genomic DNA was subjected to the determination of the number of abasic sites in the DNA using the kit according to the manufacturer’s instructions. Aldehyde reactive probe, N -aminooxymethylcarbonylhydrazin-D-biotin was used as a probe for abasic sites. The probe binds to abasic sites in the DNA through an aldehyde group present on the open ring form of the abasic sites. Finally, the number of abasic sites per 100,000 bp in the DNA was calculated using the calibration curve of standard DNA provided in the kit as a supplement. 2.8. Assessment of DNA single-strand breaks DNA single breaks and their repair were assessed using the procedure of Schröder et al. [12]. Briefly, Arabidopsis transformants were exposed to UV-B at room temperature for 15 min as described above. Genomic DNA was immediately isolated from the plants. Thirty ng of genomic DNA was subjected to the determination of DNA single-breaks in the DNA according to the procedure of fast micromethod DNA single-strand break assay [12]. Finally, strand scission factor (SSF) values were calculated using the following formula: SSF = log
% of double-stranded DNA in the sample % of double-stranded DNA in the control
Unwounded DNA of wild type plants was used as control. SSF values were calculated after a denaturation period of 20 min. 2.9. Detection of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidine photoproducts Cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidine photoproducts (6-4PP) were detected using an OxiSelect UV-Induced DNA Damage ELISA Combo Kit (CPD/6-4PP Quantitation, Cell Biolabs, San Diego, CA). Briefly, Arabidopsis transformants were exposed to UV-B at room temperature for 15 min as described above and immediately subjected to DNA isolation. Two g of Genomic DNA isolated from the plants was subjected to the detection of CPD-DNA or 6-4PP-DNA using the kit according to the manufacturer’s instructions. Finally, the amount of CPD-DNA or 64PP-DNA in the DNA was calculated using the calibration curve of standard denatured CPD-DNA or 6-4PP-DNA provided in the kit as a supplement. 2.10. UV protection assay T4 seeds of the transformants were plated on MS medium in a six-well plate and incubated at 22 ◦ C for 3 d in an incubator (11.8 W m−2 /16 h/d). Then, the plate was exposed to UV-B (220 W/cm2 ) for 4 h at room temperature. After UV irradiation, the plate was returned to the incubator. Viability was assessed at 14 d after re-incubation.
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Fig. 1. Identification of VvDRT100-L. (A) Deduced amino acid sequence of VvDRT100-L. The LRR N-terminal domain (LRRNT) is boxed. The nine LRRs (LRR1–LRR9) are underlined. (B) Cluster analysis. By means of the NJ method using MEGA4, a phylogenetic tree was constructed from the deduced amino acid sequences. Populus deltoides DRT100 (accession no. ABS18952). Glycine max DRT100 (accession no. NP 001241499). Medicago truncatula DRT100 (accession no. XP 003629372). Arabidopsis thaliana DRT100 (accession no. NP 187867). A. lyrata subsp. lyrata DRT100 (accession no. XP 002884914). A. thaliana DRT111 (accession no. NP 174336). A. thaliana DRT112 (accession no. NP 173459). (C) Transcription profile of VvDRT100-L in grapevine organs. Total RNA was isolated from the indicated organs and subjected to real-time RT-PCR analysis. -Actin was used as internal control. Data were calculated as gene expression relative to ˇ-actin expression. Bars indicate means ± standard deviation of triplicate experiments. skin(G), green berry skins; skin(B), black berry skins.
2.11. Statistics Data in figures are expressed as means ± standard deviations. Statistical analysis was performed with the non-parametric Dunnett’s multiple comparison test using Excel statistics software (Social Survey Research Information, Tokyo, Japan). 3. Results 3.1. Identification of gene encoding DRT100 protein in grapevine We obtained the full length of the cDNA fragment containing a coding sequence (1071 bp) from grapevine, similar to plant DRT100 protein. The deduced amino acid sequence had an LRR N-terminal domain and nine LRRs in the sequence (Fig. 1A). Cluster analysis of the deduced amino acid sequence with those of plant DRT proteins demonstrated that the sequence formed a cluster with plant DRT100 proteins, not with DRT111 and DRT112 proteins (Fig. 1B). We found two nucleotide sequences (LOC100241571, GenBank accession no. XM 002285517 and LOC100855260, GenBank accession no. XM 003633501) that were predicted to be grape DRT100-like proteins by the grape genome project, in the NCBI
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Fig. 2. Overexpression of VvDRT100-L repairs abasic sites induced by UVB irradiation in Arabidopsis plants. (A) Transcription profile of VvDRT100-L in VvDRT100-L-overexpressing plants. Three independent Arabidopsis lines overexpressing VvDRT100-L (OE1, OE2, and OE3) were generated. Total RNA was isolated from 12-day-old seedlings non-treated by UV-B and subjected to real-time RTPCR analysis. -Actin was used as internal control. Data were calculated as gene expression relative to -actin expression. Bars indicate means ± standard deviation of triplicate experiments. (B) Number of abasic sites. Twelve-day-old seedlings were exposed to UV-B and the numbers of abasic sites in the DNA isolated from the seedlings were measured. Wild type Col-0 (wild), Arabidopsis line transformed with pRI101-AN vector (pRI), and T-DNA insertion mutant drt100 (SALK 021479C) were used as control plants. Bars indicate means ± standard deviation of triplicate experiments. *p < 0.05 as compared with non-treated plant.
nucleotide database. LOC100241571 and LOC100855260 appear on chromosomes 6 and 13, respectively. Alignment analysis of the three nucleotide sequences revealed that our cloned cDNA showed a perfect match to LOC100855260. Based on these results, the cDNA sequence identified in the present study was deposited in GenBank database as V. vinifera DRT100-like protein, VvDRT100-L (GenBank accession no. JQ326308). VvDRT100-L transcripts were detected in various organs of grapevine (Fig. 1C). Green organs, including tendrils, leaves, and young berry skins, except stems, abundantly expressed VvDRT100L. In contrast, VvDRT100-L was not abundantly expressed in seeds, roots, stems, and matured black berry skins. From the organ distribution of VvDRT100-L transcripts, it is plausible that VvDRT100-L exists abundantly in ‘green’ organs frequently exposed to sunlight. 3.2. VvDRT100-L overexpression repairs DNA damage caused by UV-B irradiation To determine whether VvDRT100-L has the ability to repair DNA damage induced by UV-B irradiation, three independent Arabidopsis lines overexpressing VvDRT100-L (OE1, OE2, and OE3) were generated. All transformants overexpressed VvDRT100-L transcripts, although the transcriptional levels were different among lines (Fig. 2A).
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We also detected CPD and 6-4PP in the DNA of UV-B UVirradiated overexpressing plants. UV-B irradiation increased the amount of CPD-DNA (Fig. 4A) and 6-4PP-DNA (Fig. 4B) in the DNA of wild plants, pRI, and three VvDRT100-L-overexpressing Arabidopsis plants. The amount of CPD-DNA and 6-4PP-DNA in the DNA of OE1 with UV-B irradiation decreased compared with those of wild and pRI control plants (Fig. 4, OE1 + UV-B). However, the amounts of CPD-DNA and 6-4PP-DNA in the DNA of OE2 and OE3 with UV-B irradiation increased compared with those of wild and pRI control plants (Fig. 4, OE2 + UV-B and OE3 + UV-B). Also, there was no difference of the amounts of CPD-DNA and 6-4PP-DNA in the DNA between OE1 and drt100 (Fig. 4, drt100). Taken together, these results suggested that VvDRT100-L might not repair CPD-DNA and 6-4PP-DNA induced by UV-B irradiation, Fig. 3. Overexpression of VvDRT100-L decreases DNA single-strand breaks induced by UV-B irradiation in Arabidopsis plants. Twelve-day-old seedlings were exposed to UV-B and the frequency of DNA single-strand breaks (SSF value) in the DNA isolated from the seedlings was determined. Unwounded DNA of wild type plants was used as control. Bars indicate means ± standard deviation of four independent experiments with two seedlings. *p < 0.05 as compared with pRI.
UV-B irradiation increased the number of abasic sites in the DNA of Arabidopsis wild plants and pRI101-AN vector transformed plants (Fig. 2B, wild and pRI). In all of the three VvDRT100-Loverexpressing Arabidopsis plants, the numbers of abasic sites in the DNA without UV-B irradiation were less than those of control plants, although the difference was minimal compared with control plants (Fig. 2B, OE1, OE2, and OE3). The numbers of abasic sites in the DNA of the overexpressing plants were decreased by UV-B irradiation (Fig. 2B, OE1 + UV-B, OE2 + UV-B, and OE3 + UV-B). In the T-DNA insertion mutant drt100, UV-B irradiation markedly increased the number of abasic sites in the DNA (Fig. 2, drt100). DNA single-strand breaks are also generated during excision repair of abasic sites from the DNA damaged by UV irradiation [13]. The frequencies of DNA single-strand breaks in all of the overexpressing plants decreased under UV-B irradiation compared with that of pRI (Fig. 3). In the DNA of drt100, the breaks by UV-B irradiation increased compared with that of pRI.
3.3. VvDRT100-L overexpression confers tolerance to lethal UV-B irradiation in plants To evaluate whether VvDRT100-L protects plant cells from UV damage induced by lethal UV-B irradiation, we performed UV protection assay using the three VvDRT100-L-overexpressing lines. Lethal UV-B irradiation resulted in withered hypocotyls and leaves of drt100 mutant (Fig. 5). The growth of wild plants and pRI101AN vector transformed plants were also suppressed by lethal UV-B irradiation. Two of VvDRT100-L overexpressing seedlings (OE2 and OE3), especially OE2, were noticeably healthy even after lethal UVB irradiation. However, OE1 show only a modest toleration against lethal UV irradiation. 4. Discussion Plants require sunlight for photosynthesis. As UV-B is predominantly absorbed by stratospheric ozone, a very small dose reaches plants on Earth. However, due to the rapid depletion of the ozone layer over the past two decades, a large dose of UV-B has reached the Earth, resulting in unavoidable damage to plants exposed to it [14]. To avoid the deleterious effects of UV-B on macromolecules in plant cells, plants produce a large number of UV-B-absorbing
Fig. 4. Overexpression of VvDRT100-L has no effect on the repair of (A) CPD-DNA and (B) 6-4PP-DNA induced by UV-B irradiation in Arabidopsis plants. Twelve-day-old seedlings were exposed to UV-B and the amounts of CPD-DNA or 6-4PP-DNA in the DNA isolated from the seedlings were measured. Wild type Col-0 (wild), Arabidopsis line transformed with pRI101-AN vector (pRI), and T-DNA insertion mutant drt100 were used as control plants. Bars indicate means ± standard deviation of four independent experiments.
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Fig. 5. Overexpression of VvDRT100-L confers tolerance to lethal UV-B irradiation in Arabidopsis plants. Three-day-old seedlings after plating were exposed to UV-B for 4 h at room temperature. Viability was assessed at 14 d after re-incubation. The results were reproducible in three independent experiments. Scale bars = 1 cm.
phenolic compounds. Arabidopsis mutant uvt1 acquired tolerance to lethal UV-B irradiation by increasing the number of UVabsorbing phenolic pigments [15]. UV-B irradiation also caused DNA damage in plant cells. In the present study, we demonstrated that UV-B irradiation of plants led to DNA damage through the formation of abasic sites (Fig. 2), the generation of DNA single-strand breaks (Fig. 3), and the production of CPD and 6-4PP (Fig. 4), in the DNA. Homologous recombination by the RecA-mediated DNA repair machinery is the most well characterized DNA damage repair system [4]. The present study is the first to show that plant DRT100, a member of plant RecA proteins, plays important roles in DNA damage repair and toleration against UV-B irradiation. Only a few reports related to plant DRT protein family have been published [4,5,7]. To our knowledge, Pang et al. [5] were the first to identify the Arabidopsis DRT100 gene; they showed that DRT100 partially complemented the growth phenotype of E. coli recA mutants under UV irradiation. The overexpression of VvDRT100-L in Arabidopsis plants enhanced the activity of the DNA damage repair system under UV-B irradiation (Figs. 2 and 3). Reciprocally, the drt100 mutant, in which DRT100 transcripts were reduced by 24-fold compared with the wild type [4], lost its DNA damage repair activity and showed an increase in the number of abasic sites and the frequency of DNA singlestrand breaks in the DNA under UV-B irradiation. Accordingly, we estimated that VvDRT100-L might play an important role in repairing DNA damage induced by UV-B irradiation. As the oxidative damage of DNA led to the formation of abasic sites in the DNA [10], UV-B irradiation might induce the production of reactive oxygen species in plant cells [16]. This oxidative attack on deoxyriboses released free bases from DNA, resulting in strand breakage and the generation of abasic sites [10]. Based on this biochemical reaction of DNA induced by UV-B irradiation, the overexpressed VvDRT100-L protein may function as one of the master proteins in homologous recombination, which is important for the repair of strand DNA breakage, similarly to RecA in E. coli [17]. The numbers of abasic sites in the DNA of VvDRT100-Loverexpressing plants were decreased by UV-B irradiation as compared with non-irradiated plants (Fig. 2). UV-B irradiation induced the formation of abasic sites (Fig. 2), the generation of
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DNA single-strand breaks (Fig. 3), and the production of CPD and 6-4PP (Fig. 4), in the DNA of Arabidopsis plant. In plants, direct DNA repair machinery, such as nucleotide excision repair and homologous recombination, is activated to maintain genome integrity under UV stress [18]. However, VvDRT100-L did not repair CPD-DNA and 6-4PP-DNA induced by UV-B irradiation. Therefore, VvDRT100-L might specifically play a role in the repair/reduction of abasic sites and DNA single-strand breaks in plants. Taken together, these results raises a hypothesis that VvDRT100 interacts with UVB-induced or activated DNA repair machinery, resulting in the decrease of abasic sites and the frequency of DNA single-strand breaks in the overexpressing plants. Future investigation of the function of VvDRT100-L in vitro or in vivo is required and may provide new information on the DNA damage repair system in plants exposed to UV-B. The overexpression of VvDRT100-L resulted in the acquisition of tolerance to lethal UV-B irradiation in Arabidopsis (Fig. 5, OE2 and OE3). In contrast to the repair of DNA damage induced by short term UV-B irradiation, a great number of VvDRT100-L might be required for the acquisition of tolerance to lethal UV-B irradiation. This incompatibility may suggest the multiple functions of plant DRT100 in UV damage repair/toleration machinery of plants. Since it has been not reported that plant DRT100 confers UV damage tolerance in plants, we need further experiment focusing on both the cell death pathway induced by lethal UV-B irradiation and the mechanism involved in the acquisition of UV-B damage tolerance in plants. Conflicts of interest statement The authors declare that they have no conflicts of interest. Acknowledgement We are grateful to Mr. Keisuke Sugiyama of the University of Yamanashi for helpful discussions. References [1] S. Vidal, L. Francis, S. Guyot, N. Marnet, M. Kwiatkowski, R. Gawel, V. Cheynier, E.J. Waters, The mouth-feel properties of grape and apple proanthocyanidins in wine-like medium, J. Sci. Food Agric. 83 (2003) 564–573. ´ M. Riederer, E.E. Pfündel, UV screening by phenolics in [2] C.A. Kolb, J. Kopecky, berries of grapevine (Vitis vinifera), Funct. Plant Biol. 30 (2003) 1177–1186. [3] H. Frohnmeyer, D. Staiger, Ultraviolet-B radiation-mediated responses in plants. Balancing damage and protection, Plant Physiol. 133 (2003) 1420– 1428. [4] B.A. Rowan, D.J. Oldenburg, A.J. Bendich, RecA maintains the integrity of chloroplast DNA molecules in Arabidopsis, J. Exp. Bot. 61 (2010) 2575–2588. [5] Q. Pang, J.B. Hays, I. Rajagopal, A plant cDNA that partially complements Escherichia coli recA mutations predicts a polypeptide not strongly homologous to RecA proteins, Proc. Natl. Acad. Sci. U.S.A. 89 (1992) 8073–8077. [6] V. Shedge, M. Arrieta-Montiel, A.C. Christensen, S.A. Mackenzie, Plant mitochondrial recombination surveillance requires unusual RecA and MutS homologs, Plant Cell 19 (2007) 1251–1264. [7] Q. Pang, J.B. Hays, I. Rajagopal, T.S. Schaefer, Selection of Arabidopsis cDNAs that partially correct phenotypes of Escherichia coli DNA-damage-sensitive mutants and analysis of two plant cDNAs that appear to express UV-specific dark repair activities, Plant Mol. Biol. 22 (1993) 411–426. [8] M. Kohno, H. Takato, H. Horiuchi, K. Fujita, S. Suzuki, Auxin-nonresponsive grape Aux/IAA19 is a positive regulator of plant growth, Mol. Biol. Rep. 39 (2012) 911–917. [9] K.J.D. Tamura, M. Nei, S. Kumar, MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0, Mol. Biol. Evol. 24 (2007) 1596–1599. [10] S.J. Clough, A.F. Bent, Floral dip: a simplified method for Agrobacteriummediated transformation of Arabidopsis thaliana, Plant J. 16 (1998) 735– 743. [11] H.A. Dahlmann, V.G. Vaidyanathan, S.J. Sturla, Investigating the biochemical impact of DNA damage with structure-based probes: abasic sites, photodimers, alkylation adducts, and oxidative lesions, Biochemistry 48 (2009) 9347– 9359. [12] H.C. Schröder, R. Batel, H. Schwertner, O. Boreiko, W.E.G. Müller, Fast micromethod DNA single-strand-break assay, Methods Mol. Biol. 314 (2006) 287–305.
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