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

Planta volume 239, pages1101–1111(2014)Cite this article

Abstract

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.

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Abbreviations

Cd:

Cadmium

CPD:

Cyclopyrimidine dimer

ELISA:

Enzyme-linked immunosorbent assay

EndoIII:

Endonuclease III

EV:

Control transgenic plant transformed with empty vector (pBI121)

FOX:

Full-length cDNA over-expressor gene hunting system

MMC:

Mitomycin C

MS:

Murashige–Skoog

NER:

Nucleotide excision repair

REX:

Required for excision

TFB5:

RNA polymerase II transcription factor B5

TFIIH:

DNA repair/basal transcription factor IIH transcription factor IIH

UVB:

Ultraviolet B

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Acknowledgments

Drs. J. A. Ranish (Institute of Systems Biology, Seattle, WA, USA), N.-H. Chua (Rockefeller Univ, New York, USA) are acknowledged for kindly providing yeast strains and GFP plasmid, respectively. Ms. H. Yamaguchi (Tohoku University, Sendai, Japan) is acknowledged for her technical assistance. This study was supported by the Japan Society for the Promotion of Science (JSPS) scientific grants (24·7609 to SK; 24241028 & 25120702 to JH). The Saito Gratitude Foundation is gratefully acknowledged for supporting SK.

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Affiliations

  1. Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba, Sendai, Miyagi, 980-8577, Japan

    Shuta Kunihiro, Hikaru Kowata, Jun Hidema & Tomonobu Kusano

  2. RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan

    Youichi Kondou, Shinya Takahashi & Minami Matsui

  3. Biodiversity and Climate Research Center (BiK-F), Georg-Voigt-Strasse 14-16, 60325, Frankfurt, Germany

    Thomas Berberich

  4. Faculty of Bioresource Sciences, Akita Prefectural University, 241-7 Kaidobata Nishi, Akita, 010-1095, Japan

    Shohab Youssefian

Authors
  1. Shuta Kunihiro
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  2. Hikaru Kowata
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  9. Tomonobu Kusano
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Correspondence to Tomonobu Kusano.

Electronic supplementary material

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Supplementary material 1 (TIFF 540 kb) Table S1 Primers used in this study

Supplementary material 2 (TIFF 726 kb) Table S2 Functional annotation of the transgenes contained in the selected Cd-tolerant FOX lines

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

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)

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)

a

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 d uvr2-1. a and c White light conditions. b and d Red light conditions

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Kunihiro, S., Kowata, H., Kondou, Y. et al. 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. Planta 239, 1101–1111 (2014). https://doi.org/10.1007/s00425-014-2042-1

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Keywords

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