Gamma rays are the most frequently used ionizing radiation in plant mutagenesis; however, few studies are available on the characteristics of mutations at a genome-wide level. Here, we quantitatively and qualitatively characterized the mutations induced by acute/chronic gamma ray irradiation in Arabidopsis. The data were then compared with those previously obtained for carbon ion irradiation. In the acute irradiation of dry seeds at the same effective survival dose, gamma rays and carbon ions differed substantially, with the former inducing a significantly greater number of total mutation events, while the number of gene-affecting mutation events did not differ between the treatments. This may result from the gamma rays predominantly inducing single-base substitutions, while carbon ions frequently induced deletions ≥2 bp. Mutation accumulation lines prepared by chronic gamma irradiation with 100–500 mGy/h in five successive generations showed higher mutation frequencies per dose compared with acute irradiation of dry seeds. Chronic gamma ray irradiation may induce larger genetic changes compared with acute gamma ray irradiation. In addition, the transition/transversion ratio decreased as the dose rate increased, suggesting that plants responded to very low dose rates of gamma rays (∼1 mGy/h), even though the overall mutation frequency did not increase. These data will aid our understanding of the effects of radiation types and be useful in selecting suitable radiation treatments for mutagenesis.

Ionizing radiation including heavy-ion beams has been widely used in mutation breeding. Dry seeds, seedlings, and cultured tissues are often used for mutagenesis; however, little is known about the differences in induced mutations among them. Here, we examined the characteristics of mutations using randomly chosen Arabidopsis M₂ plants derived from dry seeds and seedlings irradiated with carbon ions. The mutation frequency was 1.4–1.9 times higher in dry-seed irradiation than in seedling irradiation. This difference was mainly due to the three-times higher frequency of insertions and deletions (InDels) in dry-seed irradiation than in seedling irradiation. This difference increased the proportion of mutations predicted to affect gene function among all mutations identified by whole genome re-sequencing. Our results demonstrate that the physiological status of plant tissue greatly affects the characteristics of mutations induced by ionizing radiation, and that dry seeds are more suitable materials than seedlings for inducing loss-of-function mutations. The results also showed that single base deletions often occurred in homopolymeric sequences, while InDels larger than 2–3 bp often occurred in or near polynucleotide-repeat or microhomologous sequences. Interestingly, microhomology was less commonly found around large deletions (≥50 bp), suggesting that the rejoining process differs depending on the deletion size.

To investigate the involvement of the non-homologous end joining (NHEJ) pathway in plant mutagenesis by ionizing radiation, we conducted a genome-wide characterization of the mutations induced by gamma rays in NHEJ-deficient Arabidopsis mutants (AtKu70−/− and AtLig4−/−). Although both mutants were more sensitive to gamma rays than the wild-type control, the AtKu70−/− mutant was slightly more sensitive than the AtLig4−/− mutant. Single-base substitutions (SBSs) were the predominant mutations in the wild-type control, whereas deletions (≥2 bp) and complex-type mutations [i.e. more than two SBSs or short insertion and deletions (InDels) separated by fewer than 10 bp] were frequently induced in the mutants. Single-base deletions were the most frequent deletions in the wild-type control, whereas the most common deletions in the mutants were 11–30 bp. The apparent microhomology at the rejoined sites of deletions peaked at 2 bp in the wild-type control, but was 3–4 bp in the mutants. This suggests the involvement of alternative end joining and single-strand annealing pathways involving increased microhomology for rejoining DNA ends. Complex-type mutations comprising short InDels were frequently detected in the mutants, but not in the wild-type control. Accordingly, NHEJ is more precise than the backup pathways, and is the main pathway for rejoining the broken DNA ends induced by ionizing radiation in plants.

The molecular nature of mutations induced by ionizing radiation and chemical mutagens in plants is becoming clearer owing to the availability of high-throughput DNA sequencing technology. However, few studies have compared the induced mutations between different radiation qualities and between different irradiated materials with the same analysis method. To compare mutation induction between dry-seeds and seedlings irradiated with carbon ions and gamma rays in Arabidopsis, in this study we detected the mutations induced by seedling irradiation with gamma rays and analyzed the data together with data previously obtained for the other irradiation treatments. Mutation frequency at the equivalent dose for survival reduction was higher with gamma rays than with carbon ions, and was higher with dry-seed irradiation than with seedling irradiation. Carbon ions induced a higher frequency of deletions (2−99 bp) than gamma rays in the case of dry-seed irradiation, but this difference was less evident in the case of seedling irradiation. This result supported the inference that dry-seed irradiation under a lower water content more clearly reflects the difference in radiation quality. However, the ratio of rearrangements (inversions, translocations, and deletions larger than 100 bp), which are considered to be derived from the rejoining of two distantly located DNA breaks, was significantly higher with carbon ions than gamma rays irrespective of the irradiated material. This finding suggested that high-linear energy transfer radiation induced closely located DNA damage, irrespective of the water content of the material, that could lead to the generation of rearrangements. Taken together, the results provide an overall picture of radiation-induced mutation in Arabidopsis and will be useful for selection of a suitable radiation treatment for mutagenesis.

Mutations caused by ion beams have been well-studied in plants, including ornamental flowers, rice, and algae. It has been shown that ion beams have several significantly interesting features, such as a high biological effect and unique mutation spectrum, which is in contrast to low linear energy transfer (LET) radiation such as gamma rays. In this study, the effects of double strand breaks and 8-oxo-2′-deoxyguanosine (8-oxodG) caused by ion-beam irradiation were examined. We irradiated repair-gene-inactive strains rad52, ogg1, and msh2 using carbon ion beams, analyzed the lethality and mutagenicity, and characterized the mutations. High-LET carbon ion-beam radiation was found to cause oxidative base damage, such as 8-oxodG, which can lead to mutations. The present observations suggested that nucleotide incorporation of oxidative damage gave only a limited effect on cell viability and genome fidelity. The ion-beam mutations occurred predominantly in 5′-ACA-3′ sequences, and we detected a hotspot at around +79 to +98 in URA3 in wild-type and mutant lines, suggesting the presence of a mutation-susceptible region.

The unique energy transfer characteristics of swift cluster ions have attracted the attention of many researchers working on the analysis or processing of material surfaces, but the effects on living organisms remain unclear. We irradiated B. subtilis spores with monomer and cluster proton beams and examined their lethality; the 2 MeV H₂⁺ shows a clearly lower lethality than 340 keV H⁺, even though both have a comparable linear energy transfer. The 2 MeV H₂⁺ dissociates into a pair of 1 MeV H⁺ by losing the bonding electrons at the target surface. The estimated internuclear distance and the radial dose distribution suggest that the spread of deposited total energy over two areas separated by just several nanometers greatly diminishes beam lethality and that the energy density in the very center of the trajectory, possibly within a 1 nm radius, has a great impact on lethality. We also performed a whole genome resequencing of the surviving colonies to compare the molecular nature of mutations but failed to find a clear difference in overall characteristics. Our results suggest that cluster beams may be a useful tool for understanding biological effects of high linear energy transfer radiation.