, Volume 239, Issue 5, pp 1101–1111 | Cite as

Overexpression of rice OsREX1-S, encoding a putative component of the core general transcription and DNA repair factor IIH, renders plant cells tolerant to cadmium- and UV-induced damage by enhancing DNA excision repair

  • Shuta Kunihiro
  • Hikaru Kowata
  • Youichi Kondou
  • Shinya Takahashi
  • Minami Matsui
  • Thomas Berberich
  • Shohab Youssefian
  • Jun Hidema
  • Tomonobu Kusano
Original Article


Screening of 40,000 Arabidopsis FOX (Full-length cDNA Over-eXpressor gene hunting system) lines expressing rice full-length cDNAs brings us to identify four cadmium (Cd)-tolerant lines, one of which carried OsREX1-S as a transgene. OsREX1-S shows the highest levels of identity to Chlamydomonas reinhardtii REX1-S (referred to as CrREX1-S, in which REX denotes Required for Excision) and to yeast and human TFB5s (RNA polymerase II transcription factor B5), both of which are components of the general transcription and DNA repair factor, TFIIH. Transient expression of OsREX1-S consistently localized the protein to the nucleus of onion cells. The newly generated transgenic Arabidopsis plants expressing OsREX1-S reproducibly displayed enhanced Cd tolerance, confirming that the Cd-tolerance of the initial identified line was conferred solely by OsREX1-S expression. Furthermore, transgenic Arabidopsis plants expressing OsREX1-S exhibited ultraviolet-B (UVB) tolerance by reducing the amounts of cyclobutane pyrimidine dimers produced by UVB radiation. Moreover, those transgenic OsREX1-S Arabidopsis plants became resistant to bleomycin (an inducer of DNA strand break) and mitomycin C (DNA intercalating activity), compared to wild type. Our results indicate that OsREX1-S renders host plants tolerant to Cd, UVB radiation, bleomycin and mitomycin C through the enhanced DNA excision repair.


Arabidopsis Cd-tolerance DNA excision repair FOX-hunting system Rice cDNA UVB tolerance 





Cyclopyrimidine dimer


Enzyme-linked immunosorbent assay


Endonuclease III


Control transgenic plant transformed with empty vector (pBI121)


Full-length cDNA over-expressor gene hunting system


Mitomycin C




Nucleotide excision repair


Required for excision


RNA polymerase II transcription factor B5


DNA repair/basal transcription factor IIH transcription factor IIH


Ultraviolet B

Supplementary material

425_2014_2042_MOESM1_ESM.tif (540 kb)
Supplementary material 1 (TIFF 540 kb) Table S1 Primers used in this study
425_2014_2042_MOESM2_ESM.tif (727 kb)
Supplementary material 2 (TIFF 726 kb) Table S2 Functional annotation of the transgenes contained in the selected Cd-tolerant FOX lines
425_2014_2042_MOESM3_ESM.tif (1.1 mb)
Supplementary material 3 (TIFF 1104 kb) Fig. S1 Typical first-screening result using rice FOX Arabidopsis mutant lines. A candidate Cd-tolerant line is marked by the red arrow
425_2014_2042_MOESM4_ESM.tif (1.8 mb)
Supplementary material 4 (TIFF 1793 kb) Fig. S2 Amino acid alignment of OsREX1-S and homologous proteins from other plants, including Chlamydomonas. Accession numbers are follows: OsREX1-S (Oryza sativa, NM_001066603), ZmREX1-S (Zea mays, EU963749), SbREX1-S (Sorghum bicolor, XP_002440981), HvREX1-S (Hordeum vulgare, BSK07005), PtREX1-S (Populus trichocarpa, XP_002329886), AtREX1-S (Arabidopsis thaliana, BT010796), CrREX1-S (Chlamydomonas reinhardtii, AAP12520)
425_2014_2042_MOESM5_ESM.tif (1.7 mb)
Supplementary material 5 (TIFF 1697 kb) Fig. S3 Analysis of the DNA in Cd-treated control transgenic plants and the transgenic plants expressing OsREX1-S. The seeds of control transgenic Arabidopsis (EV), REX1-1 and REX1-2 transgenic Arabidopsis plants were germinated on either control MGRL plates containing 1% gellan gum or the same media containing 125 μM CdCl2 for 1 week. Then, the DNAs prepared from them were incubated with or without endonuclease III (EndoIII) for 3 h and analyzed by alkali-agarose gel electrophoresis as described by Hidema et al. (2000)
425_2014_2042_MOESM6_ESM.tif (1 mb)
Supplementary material 6 (TIFF 1040 kb) Fig. S4 Validation of our root-bending assay using the uvr2-1 mutant after UVB irradiation. One-week-old control (Ler) and uvr2-1 mutant plants were UV-irradiated (0, 0.5 kJ, 1 kJ or 2 kJ), and then incubated further under either normal white light or red light conditions. Root lengths after UVB irradiation were measured using a ruler. a and b Control (Ler). c and duvr2-1. a and c White light conditions. b and d Red light conditions


  1. Banerjee S, Flores-Rozas H (2005) Cadmium inhibits mismatch repair by blocking the ATPase activity of the MSH2-MSH6 complex. Nucleic Acids Res 33:1410–1419PubMedCentralCrossRefPubMedGoogle Scholar
  2. Brennan RJ, Schiestl RH (1996) Cadmium is an inducer of oxidative stress in yeast. Mutant Res 356:171–178CrossRefGoogle Scholar
  3. Cenkci B, Petersen JL, Small GD (2003) REX1, a novel gene required for DNA repair. J Biol Chem 278:22574–22577CrossRefPubMedGoogle Scholar
  4. Choi YE, Harada E, Wada M, Tsuboi H, Morita Y, Kusano T, Sano T (2001) Detoxification of cadmium in tobacco plants: formation and active excretion of crystals containing cadmium and calcium through trichomes. Planta 213:45–50CrossRefPubMedGoogle Scholar
  5. Clemens S (2006) Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants. Biochimie 88:1707–1719CrossRefPubMedGoogle Scholar
  6. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743CrossRefPubMedGoogle Scholar
  7. Compe E, Egly JM (2012) TFIIH: when transcription met DNA repair. Nature Rev Mol Cell Biol 13:343–354CrossRefGoogle Scholar
  8. Costa RMA, Morgante PG, Berra CM, Nakabashi M, Bruneau D, Bouchez D, Sweder KS, Sluys M-AV, Menck CFM (2001) The participation of AtXPB1, the XPB/RAD25 homologue gene from Arabidopsis thaliana, in DNA repair and plant development. Plant J 28:385–395CrossRefPubMedGoogle Scholar
  9. Deans AJ, West SC (2011) DNA interstrand crosslink repair and cancer. Nature Rev Cancer 11:467–480CrossRefGoogle Scholar
  10. Feaver WJ, Huang W, Gileadi O, Myers L, Gustafsson CM, Kornberg RD, Friedberg EC (2000) Subunit interactions in yeast transcription/repair factors TFIIH, requirement for Tfb3 subunit in nucleotide excision repair. J Biol Chem 275:5941–5946CrossRefPubMedGoogle Scholar
  11. Fujiwara T, Yokota-Hirai M, Chino M, Komeda Y, Naito S (1992) Effects of sulfur nutrition on expression of the soybean seed storage protein genes in transgenic petunia. Plant Physiol 99:263–268PubMedCentralCrossRefPubMedGoogle Scholar
  12. Giaginis C, Gatzidou E, Theocharis S (2006) DNA repair systems as targets of cadmium toxicity. Toxicol Appl Pharm 213:282–290CrossRefGoogle Scholar
  13. Gichner T, Patkova Z, Szakova J, Znidar I, Mukherjee A (2008) DNA damage in potato plants induced by cadmium, ethyl methanesulphonate and γ-rays. Environ Exp Bot 62:113–119CrossRefGoogle Scholar
  14. Giglia-Mari G, Coin F, Ranish JA, Hoogstraten D, Theil A, Wijgers N, Jaspers NGJ, Raams A, Argentini M, van der Spek PJ et al (2004) A new, tenth subunit of TFIIH is responsible for the DNA repair syndrome trichothiodystrophy group A. Nat Genet 36:714–719CrossRefPubMedGoogle Scholar
  15. Giglia-Mari G, Miquel C, Theil AF, Mari PO, Hoogstraten D, Ng JMY, Dinant CH, Hoeijmakers JHJ, Vermeulen W (2006) Dynamic interaction of TTDA with TFIIH is stabilized by nucleotide excision repair in living cells. PLoS Biol 4:1–12Google Scholar
  16. Hidema J, Kumagai T, Sutherland BM (2000) UV radiation-sensitive Norin 1 rice contains defective cyclopyrimidine dimer photolyase. Plant Cell 12:1569–1578PubMedCentralCrossRefPubMedGoogle Scholar
  17. Higuchi M, Kondou Y, Mori M, Ichikawa T, Matsui M (2012) Characterization of rice genes using a heterologous full-length cDNA expression system. In: Dunwell JM, Wetten AC (eds) Transgenic plants: methods and protocols. Methods Mol Biol 847: 75–90. doi:10.1007/978-1-61779-558-9_8
  18. Ishikawa S, Ishimaru Y, Igura M, Kuramata M, Abe T, Senoura T, Hase Y, Arao T, Nishizawa NK, Nakanishi H (2012) Ion-beam irradiation, gene identification, and marker-assisted breeding in the development of low-cadmium rice. Proc Natl Acad Sci USA 109:19166–19171PubMedCentralCrossRefPubMedGoogle Scholar
  19. Kersten H, Rauen HM (1961) Degrasdation of deoxyribonucleic acid in Escherichia coli cells treated with mitomycin C. Nature 190:1195–1196CrossRefPubMedGoogle Scholar
  20. Kondou Y, Higuchi M, Takahashi S, Sakurai T, Ichikawa T, Kuroda H, Yoshizumi T, Tsumoto Y, Hori Y, Kawashima M et al (2009) Systematic approaches to using the FOX hunting system to identify useful rice genes. Plant J 57:883–894CrossRefPubMedGoogle Scholar
  21. Kunihiro S, Saito T, Matsuda T, Inoue M, Kuramata M, Taguchi-Shiobara F, Youssefian S, Berberich T, Kusano T (2013) Rice OsDEP1, encoding a highly cysteine-rich G protein γ subunit, confers cadmium-tolerance to yeast cells and plants. J Exp Bot 64:4517–4527PubMedCentralCrossRefPubMedGoogle Scholar
  22. Kuramata M, Masuya S, Takahashi Y, Kitagawa E, Inoue C, Ishikawa S, Youssefian S, Kusano T (2009) Novel cysteine-rich peptides from Digitaria ciliaris and Oryza sativa enhance tolerance to cadmium by limiting its cellular accumulation. Plant Cell Physiol 50:106–117CrossRefPubMedGoogle Scholar
  23. Landry LG, Stapleton AE, Lim J, Hoffman P, Hays J, Walbot V, Last RL (1997) An Arabidopsis phytolyase mutant is hypersensitive to ultraviolet-B radiation. Proc Natl Acad Sci USA 94:328–332PubMedCentralCrossRefPubMedGoogle Scholar
  24. Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigment of photosynthetic biomembranes. Method Enzymol 148:350–382CrossRefGoogle Scholar
  25. Liu Z, Hong SW, Escobar M, Vierling E, Mitchell DL, Mount DW, Hall JD (2003) Arabidopsis UVH6, a homolog of human XPD and yeast RAD3 DNA repair genes, functions in DNA repair and is essential for plant growth. Plant Physiol 132:1405–1414PubMedCentralCrossRefPubMedGoogle Scholar
  26. Mori T, Nakane M, Hattori T, Matsunaga T, Ihara M, Nikaido O (1991) Simultaneous establishment of monoclonal antibodies specific for either cyclobutane pyrimidine dimer or (6–4) photoproduct from the same mouse immunized with ultraviolet-irradiated DNA. Photochem Photobiol 54:225–232CrossRefPubMedGoogle Scholar
  27. Ranish JA, Hahn S, Lu Y, Yi EC, Li XJ, Eng J, Aebersold R (2004) Identification of TFB5, a new component of general transcription and DNA repair factor IIH. Nat Genet 36:707–713CrossRefPubMedGoogle Scholar
  28. Sakamoto AN, Lan VTT, Puripunyavanich V, Hase Y, Yokota Y, Shikazono N, Nakagawa M, Narumi I, Tanaka A (2009) A UVB-hypersensitive mutant in Arabidopsis thaliana is defective in the DNA damage response. Plant J 60:509–517CrossRefPubMedGoogle Scholar
  29. Sakurai T, Kondou Y, Akiyama K, Kurotani A, Higuchi M, Ichikawa T, Kuroda H, Kusano M, Mori M, Saitou T et al (2011) RiceFOX: a database of Arabidopsis mutant lines overexpressing rice full-length cDNA that contains a wide range of trait information to facilitate analysis of gene function. Plant Cell Physiol 52:265–273PubMedCentralCrossRefPubMedGoogle Scholar
  30. Schwerdtle T, Ebert F, Thuy C, Richter C, Mullenders LH, Hartwig A (2010) Genotoxicity of soluble and particulate cadmium compounds: impact on oxidative DNA damage and nucleotide excision repair. Chem Res Toxicol 23:432–442CrossRefPubMedGoogle Scholar
  31. Serero A, Lopes J, Nicolas A, Boiteux S (2008) Yeast genes involved in cadmium tolerance: identification of DNA replication as a target of cadmium toxicity. DNA Repair 7:1262–1275CrossRefPubMedGoogle Scholar
  32. Suzuki H, Nagai K, Yamaki H, Tanaka N, Umezawa H (1969) On the mechanism of action of bleomycin: scission of DNA strands in vitro and in vivo. J Antibiot 22:446–448CrossRefPubMedGoogle Scholar
  33. Takagi Y, Komori H, Chang WH, Hudmon A, Erdjument-Bromage H, Tempst P, Kornberg RD (2003) Revised subunit structure of yeast transcription factor IIH (TFIIH) and reconciliation with human TFIIH. J Biol Chem 278:43897–43900CrossRefPubMedGoogle Scholar
  34. Takano N, Takahashi Y, Yamamoto M, Teranishi M, Yamaguchi H, Sakamoto AN, Hase Y, Fujisawa H, Wu J, Matsumoto T et al (2013) Isolation of a novel UVB-tolerant rice mutant obtained by exposure to carbon-ion beams. J Radiat Res 54:637–648PubMedCentralCrossRefPubMedGoogle Scholar
  35. Takeuchi Y, Murakami M, Nakajima N, Kondo N, Nikaido O (1996) Induction and repair of damage to DNA in cucumber cotyledons irradiated with UV-B. Plant Cell Physiol 37:181–187CrossRefGoogle Scholar
  36. Takimoto CH, Calvo E (2008) Principles of oncologic pharmacotherapy. In: Pazdur R, Wagman LD, Camphausen KA, Hoskins WJ (eds) Cancer management: a multidisciplinary approach, 11th edn. UBM Media, London, pp 42–58Google Scholar
  37. Teranishi M, Iwamatsu Y, Hidema J, Kumagai T (2004) Ultraviolet-B sensitivities in Japanese lowland rice cultivars: cyclobutane pyrimidine dimer photolyase activity and gene mutation. Plant Cell Physiol 45:1848–1856CrossRefPubMedGoogle Scholar
  38. Tsuchiya K (1976) Epidemiological studies on cadmium in the environment in Japan: etiology of itai–itai disease. Fed Proc 35:2412–2418PubMedGoogle Scholar
  39. Vlček D, Sevcovicova A, Sviezena B, Galova E, Miadokova E (2008) Chlamydomonas reinhardtii: a convenient model system for the study of DNA repair in photoautotrophic eukaryotes. Curr Genet 53:1–22CrossRefPubMedGoogle Scholar
  40. Vonarx EJ, Tabone EK, Osmond MJ, Anderson HJ, Kunz BA (2006) Arabidopsis homologue of human transcription factor HII/nucleotide excision repair factor p44 can function in transcription and DNA repair and interacts with AtXPD. Plant J 46:512–521CrossRefPubMedGoogle Scholar
  41. Wysocki R, Tamas MJ (2010) How Saccharomyces cerevisiae copes with toxic metals and metalloids. FEMS Microbiol Rev 34:925–951CrossRefPubMedGoogle Scholar
  42. Yang SH, Berberich T, Sano H, Kusano T (2001) Specific association of transcripts of tbzF and tbz17, tobacco genes encoding basic region leucine zipper-type transcriptional activators, with guard cells of senescing leaves and/or flowers. Plant Physiol 127:23–32PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Shuta Kunihiro
    • 1
  • Hikaru Kowata
    • 1
  • Youichi Kondou
    • 2
  • Shinya Takahashi
    • 2
  • Minami Matsui
    • 2
  • Thomas Berberich
    • 3
  • Shohab Youssefian
    • 4
  • Jun Hidema
    • 1
  • Tomonobu Kusano
    • 1
  1. 1.Graduate School of Life SciencesTohoku UniversitySendaiJapan
  2. 2.RIKEN Plant Science CenterYokohamaJapan
  3. 3.Biodiversity and Climate Research Center (BiK-F)FrankfurtGermany
  4. 4.Faculty of Bioresource SciencesAkita Prefectural UniversityAkitaJapan

Personalised recommendations