Comparative analysis of seed and seedling irradiation with gamma rays and carbon ions for mutation induction in Arabidopsis

Abstract

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.

Keywords: Arabidopsis; carbon ion beam; gamma ray; mutation; structural variation.

Figure 1

Dose–response relationships for survival rate of Arabidopsis dry seeds and 7-day-old seedlings irradiated with 17.3 MeV/u carbon ions or gamma rays. Data points are the mean ± standard error of three replications with more than 25 plants. Survival curves were drawn on the basis of the single hit–multitarget theory as previously described (Hase et al., 2012). D _q is the shoulder dose. Data for gamma irradiation of seedlings were newly obtained in the present study. All other data are from our previous studies (Hase et al., 2018; Hase et al., 2020).

Figure 2

Mutations newly detected by the Manta and Lumpy algorithms. Numbers in parentheses indicate the number of mutations that correspond to each category.

Figure 3

Mutation frequency in each treatment group shown in box and whisker plots. The box presents the first and third quartiles with the center line at the median, while whiskers extend to the minimum and maximum values. The dots present outliers. The right panel presents data for homozygous mutations only and the left panel presents data for both homozygous and heterozygous mutations. Different lowercase letters above boxes in each panel indicate a significant difference (one-way ANOVA with multiple comparison test, p< 0.05).

Figure 4

Characterization of mutations induced in Arabidopsis dry seeds and 7-day-old seedlings irradiated with 17.3 MeV/u carbon ions or gamma rays. The data in this figure include both homozygous and heterozygous mutations. (A) All mutation types detected in each treatment. The proportions of rearrangements (Deletion ≥ 100 bp and structural variation [SV]) are indicated by dotted lines. Statistical comparisons of the proportions of rearrangements were performed at the equivalent dose on survival reduction. Different lowercase letters indicate a significant difference (a and b are for a comparison at dose 1 (~50% of D_q ); x and y are for a comparison at dose 2 (~75% of D_q ); Fisher’s exact test with multiple comparison correction, p< 0.05). (B) Spectra of single-base substitutions. Complementary substitutions (e.g., G:C to A:T and C:G to T:A) were merged. The T _i/T _v ratio represents the ratio of total transition to transversion events. The G:C/A:T ratio represents the ratio of G:C pairs to A:T pairs in the original nucleotides that underwent substitutions. Asterisks indicate significant differences from the A:T/G:C ratio (0.56) of the TAIR10 reference sequence (Fisher’s exact test, p< 0.05). (C) Distribution of deletion sizes.

Figure 5

Complexity of the complex-type mutation. Different lowercase letters indicate significant differences in the proportion of complex-type mutations containing two or more InDels (Fisher’s exact test with multiple comparison correction, p< 0.05). Data for dose 1 (~50% of D _q) and dose 2 (~75% of D _q) in each irradiation treatment were merged. The data in this figure include both homozygous and heterozygous mutations. Data for ku70 and ligIV mutants is from Du et al. (2020).

Figure 6

Number of protein-coding genes with non-synonymous mutations per sample. The blue bars indicate mean ± standard error in each irradiation condition. The orange bars indicate the same data but mutations that affect two more genes are excluded. The excluded deletions are listed in Supplementary Table 5 . Transposable elements, pseudogenes, and non-coding RNA were not included. SVs with unknown overall structure were also not included. Data for dose 1 (~50% of D_q ) and dose 2 (~75% of D_q ) were merged for carbon ion irradiation. Different lowercase letters indicate a significant difference (one-way ANOVA with multiple comparison test, p< 0.05.