Landscape of plastid DNA breaks in Arabidopsis during development and environmental stimulus

External and internal environmental factors can lead to DNA breaks. DNA breaks in eukaryotic nuclear genomes have been extensively studied, but the landscape of DNA breaks in endosymbiotic organelles remains poorly understood. Here, we employed DNA end tailing and sequencing (DEtail-seq) to profile DNA breaks in the Arabidopsis (Arabidopsis thaliana) plastid genome. We find that plastid DNA (ptDNA) in cotyledons and true leaves remains relatively stable during the juvenile stage but undergoes extensive breaks in older tissues. Seeds had significantly lower break levels than leaves. Notably, ribosomal DNA regions experience more breaks, with a preference for the template strand. We further investigated ptDNA break profiles in wild-type (Col-0) plants versus mutants defective in DNA repair, replication, and transcription under varying light, temperature, and photoperiod conditions. Wild-type plants maintained genome integrity under most tested conditions, but the ptDNA damage repair and replication mutants why1/3/reca1 (lacking Whirly1 (WHY1), WHY3, and RECA1), why1/3/polIb (lacking WHY1/3 and DNA polymerase IB), and atrnh1c (lacking RNase H1C) did not. Additionally, the accumulation of R-loops and reactive oxygen species (ROS) potently induces ptDNA breaks that are likely mediated by 8-oxoG modifications. Shortening the photoperiod alleviates ptDNA breaks in Col-0, with an even more pronounced effect in atrnh1c. This study provides a genome-wide view of ptDNA break dynamics and advances our understanding of damage and repair mechanisms in the organellar genome.