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Genome integrity is constantly challenged by endo- and exogenous DNA-damaging factors. The influence of genotoxic agents causes an accumulation of DNA lesions, which if not repaired, become mutations that can cause various abnormalities in a cell metabolism. The main pathway of DSB repair, which is based on non-homologous recombination, is canonical non-homologous end joining (C-NHEJ). It has been shown that this mechanism is highly conserved in both Pro- and Eukaryotes. The mechanisms that underlie DSB repair through C-NHEJ have mainly been investigated in mammalian systems, and therefore our knowledge about this process is much more limited as far as plants, and crop plants in particular, are concerned. Recent studies have demonstrated that PARP3 is an important response factor to the presence of DSB in a genome. The aims of this study were to identify the sequence of the barley PARP3 gene, to perform a mutational analysis of the sequence that was identified using the TILLING (Targeting Induced Local Lesions IN Genomes) method and to phenotype the mutants that were identified through their exposure to mutagenic treatment with the DSB-inducing chemical--bleomycin. A functional analysis led to the identification of a series of parp3 alleles. The mutants were characterized using several different approaches, including quantifying the DSB and γH2AX foci, which validated the function of the HvPARP3 gene in DSB repair in barley. The potential involvement of the HvPARP3 gene in the regulation of telomere length in barley was also analyzed.