Photorespiration is linked to DNA methylation by formate as a one-carbon source.

Photorespiration is a costly cellular process that reduces photosynthetic efficiency. While mitigating photorespiratory losses could boost crop yields, the interconnection of photorespiration with other processes is increasingly recognized. Its high carbon turnover generates mitochondrial one-carbon (C1) metabolites, including formate, but their contribution to cellular C1 metabolism has remained unclear. DNA methylation is an important epigenetic modification that depends on methyl groups provided by folate-mediated C1 metabolism. Here we show that photorespiration supplies C1 units for DNA methylation in Arabidopsis. We demonstrate that carbon from formate is incorporated into 5-methylcytosine through the C1-tetrahydrofolate synthase pathway, which operates predominantly during the day. Elevated CO2 that suppresses photorespiration alters the methylome, especially when the serine-derived C1 supply, which compensates for a blocked formate-derived supply, is compromised. These findings establish a metabolic link between photorespiration and epigenome stability and provide a framework for understanding methylome dynamics under rising CO2 levels and other environmental influences on photorespiration.