Manipulating alternative end-joining alters carbon-ion beam-induced genome mutation profiles in <i>Arabidopsis thaliana</i>

DNA double-strand breaks (DSBs) repair via POLQ-mediated alternative end-joining (Alt-EJ) is error-prone and mutagenic. However, Alt-EJ is often inhibited by classical nonhomologous end-joining (C-NHEJ) or homologous recombination, the precise impact of Alt-EJ on plant genome instability remains unclear. Here, we employed carbon-ion beam (CIB) which induce complex DSBs to bias cellular repair strategies toward Alt-EJ; additionally, a specific genetic background of C-NHEJ deficiency (lig4-4) Arabidopsis thaliana line and the POLQ-deficient (teb-3 and teb-8) were combined to further amplify the mutagenic effects of CIB mediated by Alt-EJ. The lig4-4 exhibited higher sensitivity to CIB than POLQ-deficient lines. teb-8 exhibited constitutive DNA damage response (DDR), whereas DDR in lig4-4 was strictly induced by CIB. At genome scale, lig4-4 showed substantial changes in the insertion and deletion (InDels) mutation profile, with a higher proportion and larger size of InDels as well as greater microhomology dependence than wild-type. In contrast, teb-8 showed moderate changes, including increased single-base InDels and complex mutations, but lacking > 30 bp InDels. Loss-of-function in LIG4 and POLQ resulted in a higher proportion of high-impact genome mutations than wild-type even at lower doses. These findings offered essential insights for the development of a novel repair pathway-driven heavy-ion beam mutagenesis system.