Photorespiration is linked to DNA methylation by formate as a one-carbon source [BiSulfite-seq]
DNA methylation is a key epigenetic modification regulating genome organization, stability, and gene expression. Stable DNA methylation critically relies on methyl groups provided through folate-mediated one-carbon (C1) metabolism, yet the origin and regulation of C1 supply remain elusive. Here we demonstrate that photorespiration serves as a major C1 source for DNA methylation in Arabidopsis. We show that C1 from formate, a photorespiratory byproduct, is incorporated into 5-methyl-cytosine via the reductive cytosolic folate pathway. This occurs predominantly during the day, negatively regulating serine utilization as alternative C1 source. Consequently, suppression of photorespiration under elevated CO2 levels alters the DNA methylation landscape, an effect exacerbated when regulation of C1 metabolism by the formate-dependent pathway is impaired. Thus, our findings link the fundamental metabolic process of photorespiration to epigenetic stability, highlighting how rising atmospheric CO2 levels can induce DNA methylation changes. Overall design: BS-seq analysis of Arabidopsis thaliana wild type compared to mthfd1 (m), thfs (t) single, and mt double mutants under long day (LD) conditions (8 samples in total); BS-seq analysis of mT-cr23 and mt-cr20 CRISPR-Cas9 lines (4 samples in total); BS-seq analysis of wild type and m under LD compared to short day (SD) conditions (8 samples in total); BS-seq analysis of wild type and m under high CO2 (3000 ppm) compared to control CO2 (410 ppm) conditions (8 samples in total); mRNA-seq analysis of wild type compared to mthfd1 (m), thfs (t) single, and mt double mutants under long day (LD) conditions (12 samples in total); and mRNA-seq analysis of wild type and m under high CO2 (3000 ppm) compared to control CO2 (410 ppm) conditions (12 samples in total).