Repeated drought induces a reproducible DNA methylation response associated with gene expression in Quercus lobata

Long-lived trees must continually adjust to environmental change and face sustained climatic shifts over their lifetimes. One increasingly important challenge is the rising frequency of drought caused by climate change. Environmentally responsive DNA methylation is widespread in plants, but whether it contributes to gene expression during environmental stress remains unclear, particularly in long-lived trees. Here, we integrated long read methylomes and transcriptomes from valley oak (Quercus lobata) seedlings exposed to repeated drought and well-watered treatments. Repeated drought induced a reproducible DNA methylation response that repeatedly targeted the same genomic regions despite turnover of individual methylated sites. These repeatedly targeted regions were transposable elements (TEs) located near genes. Genes adjacent to CHH-methylated TEs were enriched for core drought-response pathways, including abscisic acid signaling, osmotic adjustment and cell-wall remodeling, and remained transcriptionally activated under drought. However, higher CHH methylation levels were associated with progressively smaller transcriptional responses, suggesting that environmentally responsive DNA methylation influences how strongly drought-response genes are activated rather than simply switching them on or off. At the same time, greater CHH methylation was associated with continued repression of nearby TEs, suggesting that this response may simultaneously regulate gene activity while maintaining genome stability. Together, these findings identify a reproducible genome-regulatory response associated with repeated environmental stress in a long-lived tree. By repeatedly targeting the same genomic regions despite turnover of individual sites, this response provides a framework for how long-lived trees repeatedly adjust gene expression while maintaining genome stability during environmental change.