Characterization of gamma irradiation-induced mutations in Arabidopsis mutants deficient in non-homologous end joining

Abstract

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.

Frequency and Spectrum of Mutations Induced by Gamma Rays Revealed by Phenotype Screening and Whole-Genome Re-Sequencing in Arabidopsis thaliana

“…The molecular mutation profile at the genome scale can also be used for selecting a proper mutagen for mutation breeding and gene function analysis. Re-sequencing analysis of progeny of mutagenized plant material has provided a new perspective aimed at characterizing the mutation molecular profile of various physical mutagens at the whole-genome scale in the last 10 years [ 31 , 33 , 36 , 40 , 41 , 47 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 ]. In earlier research, only one mutant line was sequenced since it was still costly to perform NGS analysis.…”

“…For instance, Ichida detected only 3.0 inversions from 110 M 2 rice lines derived from 150 Gy carbon-ion beam irradiation [ 64 ]. Only one inversion was detected in 19 M 2 individuals of NHEJ-deficient Arabidopsis thaliana mutants (AtKu70 −/− and AtLig4 −/− ) that were irradiated with gamma rays (100 Gy) [ 60 ]. In rice, Li detected four SVs in seven M 5 plants with visible mutated traits.…”

“…C-NHEJ-mediated rejoining events usually depend on 0 to 4 bp terminal microhomology or homology, while the A-NHEJ-mediated events depend on up to 25 bp [ 3 ]. C-NHEJ-deficient Arabidopsis thaliana mutants (AtKu70 −/− and AtLig4 −/− ) used increased size of terminal microhomology at the rejoining sites generated by gamma rays [ 60 ].…”

Mutational effects of ubiquitously present gamma radiation onArabidopsis thaliana: insight into radiosensitivity in the reproductive stage

“…It is unlikely that the highly excessive number of heterozygous mutations resulted from purging homozygosity of mutations with recessive lethal effects, because the annotation analysis shows that high-impact mutations (i.e., loss-of-function gene mutations) accounted for only 7.7% of total mutations (Supplementary Table S2). Previous studies of the genetic consequences of acute/chronic radiation only during the vegetative stage showed that the heterozygous/homozygous ratio of mutations followed the Mendelian segregation ratio [e.g., 6, 12, 14, 40]. On the other hand, in this study, life-cycle chronic irradiation induced a greater number of heterogeneous mutations that should occur after gametogenesis, i.e., any reproductive stage from gamete formation to fertilization, zygote development, and then seed maturation.…”

T-DNA integration in plants requires MRE11- or TDP2-mediated removal of the 5’ bound Agrobacterium protein VirD2

“…Plant lines and growth mutants used in this study: ku70 19,42 (Col-0, SALK_123144), ku70 19,32 (Ws), ku80 19 (SALK_016627), lig4 42 (SALK_044027), tdp2-1 (SALK _043413), teb-5 9,43 (SALK_018851), mre11-2 19,29 . Double mutants were created by crossing single mutants.…”