Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 53

Contribution of multiple DNA repair pathways in Arabidopsis thaliana
dry seeds after heavy-ion-beam irradiation
K. Ishii,∗1 S. Ohbu,∗1 M. Yamada,∗1 and T. Abe∗1
Heavy-ion-beam irradiation induces DNA doublestrand break (DSB) which is critical damage to cellular proliferation. DSB is repaired by two main
pathways: homologous recombination (HR) repair,which is homology-dependent and error-free; canonicalnon-homologous end-joining (C-NHEJ), which is Kudependent and potentially error-prone and by a recently identiﬁed error-prone pathway, alternative nonhomologous end-joining (A-NHEJ). Which pathway ischosen is dependent on the phase of cell cycle and theinitiation of DNA end resection.1) HR is preferred during the S/G2 phase in the cell cycle because sister chromatids can be used as homologous templates. C-NHEJ
functions through the cell cycle and especially in G1 andG2 phase. A-NHEJ functions when the key factors ofC-NHEJ are absent, essentially in the S phase. Becausemost embryo cells in dried seeds are in the G1 phase,2)
it is possible that C-NHEJ will mainly function after
heavy-ion-beam irradiation. In this study, we irradiatedheavy-ion-beams on three diﬀerent lines: a wild type, aC-NHEJ-deﬁcient line, and a HR-deﬁcient line, and wemeasured mutation rates to reveal how each DSB-repair
pathway functions.
We obtained Arabidopsis thaliana seeds of SALK_123114 line with a T-DNA insertion in the Ku70 gene(a Ku70(−/−) mutant) and SALK_038057 line withthat in the Rad 54 gene (a Rad 54(−/−) mutant) from
Nottingham Arabidopsis Stock Centre (NASC) andused them as the C-NHEJ-deﬁcient and HR-deﬁcientlines, respectively. Dry seeds of Col-0 line (wild type),
the Ku70(−/−) mutant, and the Rad 54(−/−) mutant were irradiated with 12 C6+ (135 MeV/nucleon)
ions at doses ranging from 0 to 600 Gy. The LETof C-ion beams were controlled to 30 keV/µm. After the irradiation, ﬂowering rate (number of ﬂowering plants per total number of incubated seeds) weremeasured as survival rate, as previously described.3)
We also obtained seeds of CS16118 line with a TDNA insertion in the APG3 gene (an APG3(+/−)
mutant) from NASC. The APG3(+/−) mutant
was crossed with the Ku70(−/−) and Rad 54(−/−)
mutants to produce Ku70(−/−)APG3(+/−) andRad 54(−/−)APG3(+/−) mutants in the F2 generations, respectively. Dry seeds of the APG3(+/−) line
(regarded as a wild type), Ku70(−/−)APG3(+/−) line,and Rad 54(−/−)APG3(+/−) line were irradiated with12 6+C (135 MeV/nucleon) ions at 60 Gy. The LET ofC-ion beams was controlled to 30 keV/µm. After theirradiation, appearance ratio was measured as mutation
rate, as previously described.4)
The ﬂowering rates of un-irradiated control of the wild
∗1RIKEN Nishina Center

Fig. 1. Mutation rate after C-ion-beam irradiation at 60 Gy.
**: p < 0.01.

type, C-NHEJ-deﬁcient line, and HR-deﬁcient line were
not lower than 0.99. In the wild type, the ﬂowering
rate was 0.80 even after 600 Gy-irradiation, while in theRad 54(−/−) mutant, the rate was reduced to 0.30. In
the Ku70(−/−) mutant, there was no individual ﬂowered only after 120 Gy-irradiation. It is suggested thatboth C-NHEJ and HR pathways are involved in DNA
repair after heavy-ion-beam irradiation and the C-NHEJ
pathway has a greater contribution.
While the mutation rate of the wild type was 0.7%,
those of the HR-deﬁcient and C-NHEJ-deﬁcient lines
were signiﬁcantly (p < 0.01) higher: 2.0% and 2.9%,
respectively. It is possible that in the HR-deﬁcient lines,
DSBs usually repaired by the HR pathway are repairedby the C-NHEJ pathway. Although the diﬀerence between the HR-deﬁcient and C-NHEJ-deﬁcient lines was
not signiﬁcant, the C-NHEJ-deﬁcient line showed highermutation rate. It is suggested that when the C-NHEJ
pathway is absent, DSBs are repaired not by the errorfree HR pathway but by the error-prone A-NHEJ pathway, resulting in higher mutation rate.
In this study, it was suggested that DSBs induced indry seeds of A. thaliana by heavy-ion beams are mainly
but not entirely repaired by the C-NHEJ pathway. One
possibility is that the embryo cells in the dry seeds are
not uniform in the G1 phase. Another is that DSBs
are not completely repaired in the G1 phase but in thefollowing phase beyond the cell cycle check points.
References

1) C. S. Spampinato, Cell. Mol. Life Sci. 74, 1693 (2017).
2) R. D. de Castro, H. W. M. Hilhorst, R. Bras. Fisiol. Veg.
12, 105 (2000).
3) Y. Kazama et al., Plant Biotechnol. 25, 113 (2008).
4) Y. Kazama et al., Plant Biotechnol. 29, 441 (2012).

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