DNA damage repair in barley following double-strand breaks and its dependence on ATR

Maintaining genome integrity presents a particular challenge for plants due to their sedentary lifestyle, which disables direct avoidance of unfavorable external conditions. Additionally, plants’ metabolic processes generate reactive molecules as by-products of e.g. photosynthesis, creating internal DNA-damaging conditions. Due to this, plants have developed a unique and strictly regulated web of DNA damage responses (DDR). We initiated analysis of the DDR system in cultivated barley (Hordeum vulgare), a temperate cereal model with a large and repeat rich genome. A series of DNA damaging assays was established to describe barley plants’ phenotypic response to chemically induced DNA double-strand breaks. The efficacy of assays as a tool to be used for assessing potential new DDR barley mutants was demonstrated. DNA damage response network activation in barley was assessed by transcriptome analysis using RNA sequencing for wild type and mutant in the DDR signaling kinase ATAXIA TELANGIECTASIA MUTATED AND RAD3-RELATED (ATR). Considering the barley genome had only recently been sequenced, and it lacks the in-depth gene analysis, a list of potential DNA damage response genes in barley was compiled based on their homology with Arabidopsis genes. The comparison of transcripts in wild-type plants and atr mutants following genotoxic stress 7 days old WT (Sebastian) and mutant (hvatr.g) barley seedlings grown on solid medium in vitro were transformed for six hours into liquid medium with or without 500 μg/ml zeocin.