DNA repair under heat: DNA Polymerase λ modulates heat stress-induced mutagenesis in plants

Abstract Mutation rates can increase substantially under environmental stress, known as stress-induced mutagenesis. Specifically, heat stress has been shown to elevate mutation rates, thereby enhancing genetic variability and facilitating adaptation. However, the underlying mechanisms remain elusive in eukaryotes. Here, we investigated how heat stress affects DNA repair and induces mutations both locally and globally through CRISPR-Cas9 targeted DNA breaks and whole genome sequencing analyses in Arabidopsis thaliana. Heat stress was found to enhance CRISPR editing efficiency across all chromatin contexts, with particularly significant increases, up to 29.9-fold, in heterochromatic regions. Moreover, heat stress consistently shifts mutation outcomes toward 1-bp insertions regardless of chromatin states. We identified a heat-inducible, error-prone DNA polymerase, Polλ, as the key mediator of mutation profile changes. When extending our investigation from targeted mutations to genome-wide effects, we found that increases in global mutation rates under heat stress are also dependent on Polλ. Single-cell transcriptomic analysis further demonstrated that Polλ expression is tightly regulated and cell-type specific, with the highest expression levels in central zone meristematic cells. Together, these findings provide practical applications for improving editing efficiency in heterochromatic regions and fundamental insights into heat-induced mutagenesis, establishing Polλ as a crucial mediator of stress-induced genetic variation in plants. Competing Interest Statement F.Z. holds stock options and is a founding advisor to Viridian Genetics, which has licensed IPs from the University of Minnesota. The University of Minnesota holds equity and right to royalties under the license agreement. These interests have been reviewed and managed by the University of Minnesota in accordance with its Conflict of Interest policies. Data Availability The Data can be found at NCBI Sequence Read Archive under PRJNA1253746. All CRISPResso and WGS code can be found here: https://github.com/ZhangLab-UMN/Wootan_DNARepair_manuscript/tree/main