The seeds of Arabidopsis thaliana var. Landsberg erecta were provided by Kumho Life Science Laboratory (Kwangju Metropolitan City, Republic of Korea) and used as the plant material. Proton beams were irradiated using a proton linear accelerator (TR103) at the Korea Multi-purpose Accelerator Complex (KOMAC, Gyeongju, Korea), Korea Atomic Energy Research Institute. Dried Arabidopsis seeds were irradiated with the 100 MeV proton beam [LET = 0.7306 keV/μm] at 113.7, 190.3, 280.6, 393.4, 493.4, 574.0, 682.1, 786.9, 994.9, and 1,188.4 Gy. Gamma-ray irradiation was performed using a ⁶⁰Co gamma-irradiator (150 TBq capacity; Atomic Energy of Canada Limited, Ottawa, Canada) at the Advanced Radiation Technology Institute (ARTI, Jeongeup, Korea), Korea Atomic Energy Research Institute. Dried Arabidopsis seeds were irradiated with gamma-rays [LET = 0.2 keV/μm] at 200, 400, 600, 900, 1200, and 1500 Gy.

The irradiated Arabidopsis seeds were sown in soil and cultivated at 22°C under a 16-h light/8-h dark photoperiod. Survival rate was determined 4 weeks after seeding using four replications with 50 seeds. The LD₅₀ dose (50% of the plants die) and the shoulder dose (Dq) were obtained from the survival rate–irradiation dose curve. To calculate the shoulder dose, we used a single-hit multitarget equation (Hase et al., 2012) as follows.

where D is the dose, D₀ is the dose that results in 37% survival rate, and m is the extrapolating number, which was calculated based on the least-squares method. The shoulder dose was calculated according to Hase et al. (2020) as follows.

To investigate the phenotypic mutations, the seeds of 362–490 M₁ lines were obtained from each proton beam irradiation at 493.4, 682.1, and 994.9 Gy, and gamma-irradiation at 600, 900, and 1,200 Gy. One M₂ individual from each M₁ line was grown on plates (100 × 30 mm) with 70 ml of 0.5 × MS medium (2% sucrose, 0.6% plant agar, 0.05% MES, pH 5.7). In each plate, seven M₂ individuals from different M₁ lines were grown together. Three weeks after seeding, mutants were identified by screening for color, shape, and developmental characteristics of the leaves.

Six individuals were randomly selected from the M₂ lines obtained from the proton beam irradiations at 493.4, 682.1, and 994.9 Gy, and the gamma-ray irradiation at 900 Gy. DNA was extracted from the rosette leaves of each individual plant using a cetyltrimethylammonium bromide (CTAB)-based method (Cota-Sánchez et al., 2006). DNA sequencing to obtain paired-end reads (100 bp or 150 bp) was performed for the 24 selected M₂ plants and two wild-type individuals using the Illumina HiSeq 2000 or HiSeq X Ten (Illumina, Inc., San Diego, CA, United States) platform that was commercially available at Theragen Etex (Seongnam-si, Republic of Korea). For sequence pre-processing, PCR duplicate reads were removed from the raw sequences and quality trimming was performed to obtain sequences that contained at least 25 high-quality bases (phred score ≥ 20) using the SolexaQA (v.1.13) package (Cox et al., 2010). The cleaned reads were mapped to the Ler assembly as the reference genome (GenBank accession number: GCA_001651475) using the BWA (0.6.1-r104) program (Li and Durbin, 2009) to generate BAM format files. SBSs and small InDels (<100 bp) between the reference genome and the mapped sequences from each sample were called from the BAM format files using SAMtools (0.1.16) (Li et al., 2009), an in-house script (Kim et al., 2014), and Pindel (0.2.5) (Ye et al., 2009). For SAMtools, the minimum mapping qualities for SBSs and gaps were set as 30 and 15, respectively, and the read depth range was set as 10–505. The SBSs and InDels from each sample were integrated to generate an SBS and InDel matrix to detect variations between samples. If the proportion of mutant reads was ≥80% in a mutation site, the mutation was defined as homozygous; if the proportion was ≥25 and <80%, the mutation was defined as heterozygous. To detect SVs, duplicate sequences were removed from the BAM format files using the Picard MarkDuplicates program¹. Then, BreakDancer (version 1.4.5) (Chen et al., 2009) was used to detect SVs. For BreakDancer, the default settings were used, except the minimum number of read pairs required to establish a connection was set as 3. The SVs from each sample were integrated to generate an SV matrix to detect variations between samples. The SBSs, small InDels, and SVs that were common between the wild-type individuals but were polymorphic between wild-type individuals and a single M₂ line were selected and used for the analysis. Mutations that were commonly detected in two or more M₂ lines were excluded.

The SVs detected using BreakDancer were visualized and explored using the Integrative Genome Viewer (IGV; Robinson et al., 2011). To validate the SVs, we designed primers from the flanking sequences of each SV junction and performed PCRs to determine whether the amplicons expected from each SV model were generated. The sequence of each amplicon was obtained by Sanger sequencing. BLAST was used to align the amplicon sequences from the wild type and M₂ lines that contained the SVs to determine the SV junction position and reconstruct the SV formation processes.

The survival rates of Arabidopsis were investigated 4 weeks after sowing dry seeds irradiated with 100 MeV proton beams (LET = 0.7306 keV/μm) or gamma-rays (LET = 0.2 keV/μm) at different irradiation doses (Figure 1). In both analyses, high survival rates were maintained at low doses without significant change, but they decreased rapidly at doses higher than the dose that was defined as the shoulder dose (Dq) in previous studies (Yamaguchi et al., 2009; Hase et al., 2018). The Dq calculated following Hase et al. (2012) (see section “Materials and Methods”) was 13% lower for proton beam irradiation (754 Gy) than it was for gamma-ray irradiation (860 Gy), which indicates that the negative effect of proton beams on plant survival was slightly higher than that of gamma-rays. The LD₅₀ was also slightly lower for the proton beam irradiation (1,051 Gy) than it was for the gamma-ray irradiation (1,086 Gy). Among the irradiation doses used in this study, we selected the doses that were closest to LD₅₀, Dq, and two-thirds of Dq; namely, 995, 787, and 493 Gy for proton beams and 1,200, 900, and 600 Gy for gamma-rays, respectively. The M₂ lines developed from the irradiations at these doses were used for the subsequent mutation analysis.

Phenotypes were investigated visually 3 weeks after seeding in the M₂ mutant populations developed by proton beam (493, 787, and 995 Gy) or gamma-ray (600, 900, and 1,200 Gy) irradiation. The detected mutant phenotypes were categorized as changes in leaf color, leaf shape, and plant architecture (Table 1 and Figure 2). The highest mutation rate obtained among the proton beam populations (5.52%) was slightly higher than that obtained among the gamma-ray populations (4.50%) (Table 1 and Figure 3A). The dose (787 Gy) near the shoulder dose (Dq) for proton beam irradiation produced the highest mutation frequency, whereas the highest dose (1,200 Gy) near the LD₅₀ for gamma-ray irradiation produced the highest mutation frequency. Compared to the highest mutation frequencies obtained in each radiation source, the mutation frequencies for the lowest doses that corresponded to two-thirds of Dq were more than two times lower for proton beam irradiation (2.28% in 494 Gy-irradiated population) and gamma-ray irradiation (2.04% in 600 Gy-irradiated population). The proton beams and gamma-rays also produced a different mutation spectrum. Most of the mutations (81%) found in the gamma-ray populations resulted in leaf color variations, such as albino, light green, purple, yellowish, variegated, and mottled leaves (Table 1 and Figures 2B, 3B). Proton beams produced broader and a more evenly distributed mutation spectrum than gamma-rays (Table 1 and Figures 2A, 3B). Several mutant characteristics of leaf shape (narrow, torpedo-shaped, and curled leaves) and plant architecture (extensive vegetative growth and formation of multiple main inflorescences) were detected only in the proton beam-irradiated populations. In addition, the frequencies of leaf-shape (41%) and leaf-color (49%) mutations induced by proton beam irradiation were similar between these two phenotypic categories.

Whole genome sequencing was performed for six individual M₂ plants derived from irradiations using 100 MeV proton beams at 493, 787, and 995 Gy, and gamma-rays at 900 Gy, respectively (hereafter, each M₂ plant group is referred to as “P493,” “P787,” “P995,” and “G900,” respectively). The generated sequencing reads were mapped on the 97.8% DNA region (on average) of the reference genome. The sequencing depth of the mapped reads was 46.6× on average (Supplementary Table 1). DNA mutations detected by comparative analysis between non-irradiated plants (two individuals) and each irradiated individual were classified into three main groups, SBSs, small InDels (<100 bp), and SVs, including large deletions (≥100 bp), duplications, inversions, and translocations. The average numbers of total mutated sites were 60, 73, 80, and 86 in P493, P787, P995, and G900, respectively (Figure 4). The numbers were significantly higher (p < 0.05) in P787, P995, and G900 than in P493, but no significant difference was detected among the numbers in P787, P995, and G900. This result indicates that the mutation frequency was not greatly changed when the proton beam irradiation dose was increased from Dq to LD₅₀ or when the radiation source was changed from proton beams to gamma-rays, whereas lowering the proton beam irradiation dose below Dq more affected mutation frequency. In each plant group, the proportion of mutation sites with SBSs was commonly highest (65.8–70.0%) among the three types of mutation sites. The total number of DNA regions with rejoined junctions of SVs was 26 and 28 in the six individuals of P787 and P995, respectively, but only 7 in the six individuals of both P494 and G900 (Figure 4). Therefore, the proton beam irradiation induced more SVs than the gamma-ray irradiation at doses that resulted in similar survival rates, and changing from the lower irradiation dose to Dq increased the number of SVs produced by proton beam irradiation.

The frequency of SBSs in each group was between 3.35 × 10^–11/bp and 5.09 × 10^–11/bp. The frequency of SBSs in P494 was significantly lower than those in the other three groups, and no significant difference was detected among those in the three groups (Figure 5A). The frequency of homozygous SBSs was highest in G900, followed by P995, P787, and P494 (Figure 5A). The heterozygous-to-homozygous ratio for SBSs ranged from 0.31 in P494 to 0.64 in G900. However, variations in this ratio among individuals was quite high, and therefore no significant difference (p < 0.05) from the ratio theoretically expected based on Mendelian segregation (0.5) was detected in any of the groups. The frequency of SBSs in the protein coding regions was lower than that in the non-coding regions in P995, whereas significant difference was not detected between the frequency of SBSs in the coding and non-coding regions in P493, P787, and G900 (Figure 5B). The transition-to-transversion ratio was relatively lower in the proton beam-irradiated groups (0.98–1.22) than it was the gamma-ray-irradiated group (1.25) (Figure 5C).

The frequency of small InDels (<100 bp) was between 1.62 × 10^–11/bp (P787) and 2.08 × 10^–11/bp (G900) and was not significantly different between plant groups (Figure 6A). However, the frequency of homozygous InDels was significantly higher in G900 than it was in P493 because the homozygous-to-heterozygous ratio for InDels was higher in G900 (0.45) than it was in P493 (0.13) (Figure 6A). Deletions were from 3.63 (G900) to 5.04 (P787) times more frequent than insertions (Figure 6B). By length, 1-bp deletions accounted for the largest proportion of InDels (from 39.9% in G900 to 53.9% in P787) in all the plant groups, followed by 2–9-bp deletions (between 25.2% in P787 to 33.8% in G900) (Figure 6B). Small InDels were more frequently detected in non-coding regions than in protein coding regions in P493, P995, and G900 (Figure 6C). Previous research showed that homopolymeric sequences or polynucleotide repeats were often found at the junction of small deletions (Belfield et al., 2012; Du et al., 2017; Hase et al., 2018). This is consistent with our finding that homopolymeric sequences and polynucleotide repeats were found at 27.7–41.7% and 10.8–18.1% of the junctions, respectively (Figure 6D).

We predicted SVs by in silico analysis of the short read sequences mapped on the reference genome (see section “Materials and Methods”) and validated them by PCR amplification followed by Sanger sequencing of the amplicons. Information about types, zygosity, and position in the genome was obtained in 39 rearrangement processes that were expected from the identified SVs (Table 2, Supplementary Table 3, and Supplementary Figures 1, 2). Only four and five rearrangement processes were expected in G900 and P494, respectively, whereas 15 were expected in both P787 and P995 (Table 2). Unlike heavy ion beams that easily generate complex DNA rearrangement by inducing several rearrangement events simultaneously in a short DNA region (Kazama et al., 2017), the proton beams and gamma-rays mostly induced rearrangement process that were expected to involve a single DNA rearrangement event such as deletion, inversion, translocation, and duplication. Only two SV events, SV18 and SV22 (Supplementary Table 3 and Supplementary Figure 2), involved multiple rearrangement events, which were two inversions. The 10 and 31 rearrangement events were homozygous and heterozygous, respectively. The size of the deleted or inverted DNA fragment was relatively small (<20 kb) in most of homozygous events, whereas it often exceeded 100 kb in heterozygous events (Supplementary Table 3). Inversions accounted for the largest portion (23 of 41) among the different types of rearrangement events, followed by large deletions (12 of 41) (Table 2, Supplementary Table 3). Most inversions (20 of 23) contained at least a rearrangement junction, which was expected to be formed by joining of the end sequences with microhomology between them (2–23 bp in size) (Table 3, Figure 7, and Supplementary Table 3 and Supplementary Figure 2). Although the overlapped sequences were maintained after rearrangements in three of the inversions, they were partially deleted in 17 other inversions (Figure 7C, Supplementary Table 4). In addition to deletion on the overlapped sequences, 11 inversions were associated with deletion (1–61 bp in length) on the adjacent DNA regions of the overlapped sequences (Figure 7D, Supplementary Table 4).

Mutations in the protein coding sequences were analyzed to predict the impact of DNA mutations on gene function. The total number of genes for which the sequences or lengths of the encoding proteins were changed by DNA mutations was highly variable among individuals because heterozygous large deletions in several individuals resulted in the loss of many genes at once. This hampered statistical analysis to determine significant differences between groups. Because most of the deleted fragments in the homozygous SVs were relatively short and contained fewer genes, variation in the number of genes affected by homozygous mutations among individuals was relatively small. The average number of genes affected by homozygous mutations was highest in G900 (5.33 genes) and lowest in P494 (3.33 genes) (Table 4). When genes affected by SVs were excluded, the average number of genes affected by homozygous or heterozygous mutations was higher in G900 (14.5 genes) and P787 (12.7 genes) compared with P494 (9.3 genes) (Table 4), and the average number of genes with homozygous mutations ranged from 2.7 (P493) to 4.2 (P787) and 4.8 (G900). Our results indicated that proton beam or gamma-ray irradiation at Dq induced mutations in a similar number of genes, if SVs were excluded. The proportion of genes affected by high impact mutations was 31.6% in P787 and 27.6% in G900, and 36.0% in P787 and 20.7% in G900 for only homozygous mutations, when genes affected by SVs were excluded from the analysis (Table 4). When the effect of SVs was included, the average number of genes with high impact mutations was much higher in P787 (28.2 genes) than it was in G900 (4.8 genes), but most of these genes carried heterozygous mutations and the variation among individuals was very high.

As a major component of cosmic-rays, protons may have affected the evolution of plants on Earth and could influence the growth and inheritance of plants that will be cultivated in extraterrestrial space environments in the coming space era (Girdhani et al., 2013). Protons can also be used as a novel mutagen in plant mutation breeding as other radio-active particles such as heavy ion beams and fast neutrons, which have been widely applied in breeding after characterization at the molecular level (Kim et al., 2018). We performed a comprehensive study using both phenotype and whole genome sequence analyses to characterize the mutagenic effects of proton beams in plants.

The phenotype analysis showed that proton beam irradiation resulted in a broader range of mutation spectrum than gamma-ray irradiation when the irradiation dose was Dq or LD₅₀, although the mutation frequency was not clearly different between the two irradiation sources. The reason for the broader mutation spectrum is not clear, but the higher proportion of genes carrying high impact mutations and the higher number of SVs produced in the proton beam irradiation may be causal factors. The high impact mutations, such as complete loss or truncation of genes and generation of premature stop codons, are likely to induce loss-of-function of genes that may result in visually detectable distinctive phenotypic changes. In addition, SVs, such as large deletions or inversions, not only directly affect the copy number or structure of genes but also may change the genomic landscape that is related to the transcription pattern of genes in specific genomic areas (Fransz et al., 2016). Alonge et al. (2020) showed that natural SVs led to changes in important traits in tomato (fruit size, flavor, production) by modifying gene expression levels and dosage. In peach, Guan et al. (2021) reported that a 1.67 Mb inversion lead to upregulation of PpOFP2 gene located nearby an inversion junction, and resulted in a change of fruit shape. The impact of SVs on gene expression has been studied more deeply in humans. Alterations in the genomic location of genes by inversions or translocations were suggested to cause position effects by changing the epigenetic states of DNA regions near break points (Genesio et al., 2011; Finelli et al., 2012). In such cases, the combinational effects of the multiple genes that were affected at the transcriptional levels may lead to phenotypic changes that cannot be induced by a single gene mutation. Carbon ion beam irradiation, which can also induce frequent SVs, resulted in a broader spectrum of mutant phenotypes in chrysanthemum (Tanaka et al., 2010) and carnation (Okamura et al., 2003) than that obtained with gamma-ray irradiation.

The comparative analysis between the whole genome sequences of Arabidopsis M₂ lines derived by proton beam and gamma-ray irradiation in this study and by heavy ion beam irradiation in previous studies (Kazama et al., 2017; Hase et al., 2018, 2020) enabled characterization of the mutagenic effects according to the LET or types of radiation (Figure 8). Relatively more frequent single nucleotide mutations (SBS and 1-bp InDels) were detected with low-LET radiation (gamma-rays and proton beams) than with high-LET radiation (carbon and argon beams). In addition, the transition-to-transversion ratio and proportion of InDels < 10 bp among the small InDels (<100 bp) were generally higher with the low-LET radiation. Unlike these patterns, the number of SVs was expected to be affected not only by LET but also by the type of radiation. Proton beam irradiation induced notably more frequent SVs than those produced by gamma-ray irradiation when both irradiations were performed at irradiation doses (Dq) that caused similar impairment in plant survival. The frequency of SVs induced by proton beam irradiation at Dq was higher than that produced by carbon beam irradiation at 75% Dq. These results imply that, for the induction of SVs, proton beams may have more similar characteristics with other accelerated particles with higher LET than gamma-rays with LET, which is more similar to that of proton beams. In vitro analyses by Leloup et al. (2005) and Hada and Sutherland (2006) showed that proton beam irradiation induced much higher numbers of DNA breaks than gamma-ray irradiation, even when the LET of each radiation was adjusted to be similar. Calugaru et al. (2011) also showed that proton beam irradiation always induced a higher number of clustered DNA lesions than gamma-ray irradiation, regardless of the energy of protons. In a track structure analysis, the protons deposited energy in an almost straight cylinder inducing energetic secondary electrons, whereas photon beams with the same LET produced Compton electrons that formed the scattered multidirectional stochastic tracks (Girdhani et al., 2013). This track structure of proton beams may possibly be related to their biological effects in SV induction. Taken together, protons, as accelerated particles with low-LET, have unique features in their mutagenic effects on plant DNA that are distinguished from those of heavy-ion beams with the higher LET and gamma-rays.

Although the relationship between irradiation dose and frequency of phenotypic mutations in M₂ generations has been analyzed for a wide range of doses (Yamaguchi et al., 2009; Kazama et al., 2011; Kazama et al., 2017), comparisons of DNA mutation frequency among plant samples irradiated at different doses have been reported for only a limited range of doses below the shoulder dose (Hase et al., 2018, 2020). In our whole genome sequencing analysis of plant groups irradiated with proton beams at doses near the LD₅₀, Dq, and two-thirds Dq, differences in the frequencies of SBSs, small InDels, and SVs were much smaller among groups for LD₅₀ and Dq compared with those among groups for Dq and two-thirds Dq (Figure 8). This result indicates that to increase dose in the range higher than the Dq was much less effective than to increase dose in the range below the Dq in increasing mutation frequency. Phenotypic mutation frequencies reported previously are consistent with our result. For example, the highest number of rice M₂ mutant lines per irradiated M₁ seeds was detected for gamma-ray and ion beam irradiations at the shoulder dose, which indicates that the extent of survival rate decrease in the M₁ population was not proportional to the increase in mutation frequency in the M₂ population when the irradiation dose was higher than the shoulder dose (Yamaguchi et al., 2009). Irradiation doses above a certain level greatly decreased the flowering rate of M₁ Arabidopsis plants; however, argon and iron beam irradiation doses above the same level did not increase the rate of albino plants in the M₂ population (Kazama et al., 2008). On the basis of these accumulated data, we hypothesized that there may be a threshold mutation rate that an individual plant survived after irradiation with a specific radiation can contain. Assuming that individuals with various rates of mutation are generated along a probability distribution curve by irradiation at specific dose, then only the individuals with mutation rates below a threshold will survive (Figure 9A). Therefore, increasing irradiation dose may not increase mutation rate to be higher than the thresholds, but it may increase the average mutation rate in surviving individuals to a level that is closer to the threshold mutation rate. According to this hypothesis, the distribution curve for plants irradiated at the shoulder dose will have the smallest slope at the threshold mutation rate. Because of this characteristic, the survival rates at doses higher than the shoulder dose drop sharply, but the average mutation rate among the individuals that survived does not increase that much (Figure 9B). In contrast, for doses below the shoulder dose, the ratio of mutation rate increase to survival rate decrease is much larger. This relationship between survival rate and mutation rate in our “threshold mutation rate hypothesis” fits well with the results of the present and previous studies. Further whole genome sequencing analysis of plant samples irradiated at various doses may provide more information to determine threshold mutation rates and the exact distribution pattern of irradiated samples according to the mutation rate.

We analyzed SVs induced from proton beam and gamma-ray irradiation by examining DNA rearrangement junctions at the nucleotide level. Most of the SVs had simple structures and were expected to be derived from a single DNA rearrangement event, unlike previously analyzed SVs that were induced by heavy ion beams and were mostly complex involving a series of successive rearrangements (Hirano et al., 2015; Kazama et al., 2017). The structural simplicity of the SVs in our study enabled the determination of SV types and separate analysis for each rearrangement events. Most of the DNA rearrangement events (34 of 41) involved joining of end sequences with microhomology (2–23 bp). Consistent with our results, the involvement of microhomologous sequences in DNA structural rearrangements has been reported previously in plant mutant lines developed by irradiation (Shirley et al., 1992; Shikazono et al., 2001; Kazama et al., 2017). DSBs and DNA repair by the error-prone NHEJ has been suggested as the mechanism for induction of SVs by irradiation in plants (Hirano et al., 2015). The presence or absence of microhomologous sequences and InDels around microhomologous sequences is an important factor for subclassification of the NHEJ mechanism (Mcvey and Lee, 2008). Because conservation or the presence of small InDels adjacent to a DSB can be examined in the inversion events that accounted for the largest portion of the DNA rearrangements in our study, we analyzed the rearrangement junctions in inversion events to characterize the NHEJ mechanism. Among the 23 inversions, 12 were expected to be associated with end joining via microhomologous sequences in the original DNA molecule and did not contain any deletions around DSB sites, although they did contain small deletions on the joined microhomologous sequences. This finding implies that DSBs and ligation occurred on or at the ends of microhomologous sequences in those events, which excluded the possibility that an alternative mechanism of NHEJ, microhomology-mediated end joining (MMEJ), was involved. In MMEJ the DNA regions between break points and microhomologous sequences are removed for end joining using microhomology (Ottaviani et al., 2014). The preference of using microhomology in NHEJ might be a reason for frequent presence of microhomologous sequences on the rearrangement junctions. Although microhomology is not essential for NHEJ, the use of microhomology may be dominant in the joining of DNA ends if there are microhomologous sequences in the overhangs (Gerstein and Lieber, 1993; Weinstock et al., 2007; Pannunzio et al., 2014). In yeasts, which have NHEJ systems that show high similarity to those of plants in its components, there is more reliance on terminal microhomology in NHEJ than there is in mammals (Daley and Wilson, 2005; Daley et al., 2005; Lieber, 2010). Therefore, coincidental DSBs near or at the ends of two microhomologous sequences by proton beam irradiation may lead to SV events using microhomology rather than re-joining to the original DNA without using microhomology. Besides these inversions, inversion events with very small InDels around rearrangement junctions (<10 bp; 6 of 23 inversion events) are likely to be mediated by classical NHEJ (cNHEJ). This is because deletion of a few nucleotides is often associated with cNHEJ due to the iterative and unordered actions of nuclease, polymerases, and ligase in this process (Lieber, 2010). Deletions longer than 10 bp around microhomologous sequences were found in four inversion events. MMEJ could be a mechanism for these SV. In the Arabidopsis mutant lacking KU70 (an essential component of cNHEJ), inversions induced using the CRISPR/Cas system resulted in a high proportion of rearrangement junctions joined by microhomologous sequences with deletions (10 s to > 100 nucleotides), which indicates that MMEJ can occur in plants as an alternative to cNHEJ (Schmidt et al., 2019). Therefore, we suggest that cNHEJ was responsible for most of the DNA rearrangements found in the proton beam-irradiated Arabidopsis, and MMEJ may be involved as a minor mechanism. Hirano et al. (2015) speculated that alternative NHEJ, rather than cNHEJ, might contribute to a high rate of SVs because there were frequent deletions at rearrangement junctions which were joined via microhomologous sequences in argon- and ion beam-irradiated Arabidopsis. Hase et al. (2012) showed that the sensitivity of Arabidopsis to radiation was higher in a DNA ligase IV mutant lacking the cNHEJ pathway when it was compared to wild-type Arabidopsis. The degree of the survival rate decrease according to increase of irradiation dose was much higher in low-LET carbon beam irradiation (113 keV μm^–1) than it was in high-LET carbon beam irradiation (425 keV μm^–1), which implies that the contribution of cNHEJ was higher in the repair of DNA damage by low-LET radiation than it was for high-LET radiation. The higher contribution of cNHEJ in SV generation found in irradiation of proton beams with low LET in our study may support the relationship between LET and the DNA repair mechanism reported previously.