RIKEN Accel. Prog. Rep. 48 (2015)

Ⅲ-4. Radiation Chemistry & Biology

LET-dependent effect on mutation induction DNA repair-deficient backgroundin Arabidopsis thaliana
K. Ishii,*1 Y. Kazama,*1 Y. Shirakawa,*1 S. Ohbu,*1 and T. Abe*1
A previous study on the effect of LET on inducing
mutation revealed that the most effective value of LET(LETmax) on dried seeds of Arabidopsis thaliana was 30.0
keVμm-1, within a range 22.5 to 640 keVμm-1.1) LET istherefore an important factor in mutation induction. In themutagenesis process, the DNA double-strand break (DSB)
repair system is concerned with the production of mutations.
There are two major DSB repair pathways:
non-homologous end joining (NHEJ) and homologous
recombination repair (HRR) function in eukaryotic cells.2)
NHEJ and HRR are independent pathways. HRR is arelatively error-free pathway because it utilizes thehomologous region of a sister chromatid to repair thedamaged strand, whereas the NHEJ pathway is relativelyerror-prone.
To determine whether DSB repair pathways are involvedin the LET-dependent effect on mutation induction, weintended to investigate the effect of LET in the DSB
repair-deficient background. We planned to measure themutation rates after heavy-ion beam irradiation with LET
values 22.5 and 30 keVμm-1 in the three mutant lines: 1) theHRR pathway-deficient mutant line, 2) the NHEJ
pathway-deficient mutant line, and 3) both HRR and NHEJ
pathway-deficient mutant line. Here, we report the mutation
rates of the Rad54-deficient mutant as the HRR
pathway-deficient mutant line.
Seeds of the APG3(+/-) mutant (CS16118) and theAtRad54(-/-) mutant (SALK_038057) were obtained from theArabidopsis Biological Resource Center (ABRC, OhioState University) and the European Arabidopsis StockCentre (NASC, the University of Nottingham), respectively.
The APG3(+/-) mutant carries BASTA-resistance at theAPG3-disrupted allele, and a uniformly heterozygous
population can be selected as photosynthetic andBASTA-resistant seedlings.3) The uniformly heterozygous
population facilitates investigation of the mutation
frequency in the irradiated (M1) generation by calculating
the proportion of the number of plants with white sectors ontrue leaves to that of total plants with true leaves (Fig. 1).
The AtRad54(-/-) mutant carries a kanamycin resistance. The
APG3(+/-) plants were crossed with the AtRad54(-/-) plants.
The F1 seeds were germinated in the presence of BASTA (2μg/mL) and kanamycin (50 μg/mL), and the germinatedplants were replanted to pots. F2 seeds were collected fromthe self-pollinated F1 plants. In the F2 generation, thephotosynthetic and both BASTA- and kanamycin-resistant
plants were screened. The second screening of the
APG3(+/-)/AtRad54(-/-) plants were conducted by PCR. Asufficient number of seeds were collected from progenies of*1

RIKEN Nishina Center

the identified APG3(+/-)/AtRad54(-/-) plants.
Heavy-ion beam irradiation was conducted as previously
described3) with some modifications. The seeds of the
APG3(+/-)/AtRad54(-/-) and APG3(+/-)/AtRad54(+/+) plants wereirradiated with 12C6+ ions with LETs of 22.5 or 30.0
keVμm-1 at a dose of 300 Gy.
The mutation frequencies were obtained as previously
described.3) The mutation frequencies of AtRad54(+/+) plants
were 3.0 and 6.6% when the LET values were 22.5 and 30.0
keVμm-1, respectively, and they are significantly different(Table 1; p<0.05 with chi-square test). The mutation
frequency of 22.5 keVμm-1-irradiated AtRad54(-/-) plants
was 5.6%, which was at the same level as that of30.0-keVμm-1 irradiated control (p≥0.05). It is assumed that
because the HRR pathway is disabled, the error-prone
NHEJ pathway mainly functioned to repair DSB. Themutation frequency of 30.0-keVμm-1 irradiated AtRad54(-/-)
plants, however, was still 6.8%, which was at the same level
as that of the control (p≥0.05). This result proposed ahypothesis: in the case of the 30.0-keVμm-1 irradiation, in
contrast to the 22.5-keVμm-1 irradiation, DSBs occur
beyond the capacity of the HRR pathway functions and arerepaired mainly by the NHEJ pathway, leading to a highmutation frequency. Further analysis on other DNA repair
gene-deficient mutants is in progress.

Fig. 1. Sector mutation caused by heavy-ion beamirradiation on the APG3(+/-) mutant. (A) A plant showing
sector mutation. The leaf exhibiting mutation is indicatedby the white arrowhead. (B) A plant not showing abnormal
phenotype. Bars = 2 mm.
Table 1. LET-dependent effect on inducing mutation.
Mutation frequency (%)
LET (keVμm-1)
AtRad54(-/-)
AtRad54(+/+)
22.5
5.6 (916)
3.0* (2,241)30.0
6.8 (857)
6.6* (2,118)
Numbers in parentheses indicate numbers of samples.
*Kazama et al. (2012).
References1) Y. Kazama et al.: Plant Biotechnol. 25: 113-117 (2008).
2) L. H. Thompson: Mutat. Res. 751: 158-246 (2012).
3) Y. Kazama et al.: Plant Biotechnol. 29: 441-445 (2012).

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