Genomic context‐dependent roles of 5‐hydroxymethylcytosine in regulating gene expression during rice drought response

SUMMARY DNA methylation (5‐methylcytosine, 5mC) is a key epigenetic regulator of genome stability and stress adaptation in plants. However, the functional role of its oxidative derivative, 5‐hydroxymethylcytosine (5hmC), remains poorly understood in plant systems, largely due to its low abundance and unresolved enzymatic origins. Here, we integrated ACE‐seq (APOBEC‐coupled epigenetic sequencing) with an optimized Tn5mC‐seq (transposase‐based library preparation in the context of whole‐genome bisulfite sequencing, WGBS) approach to generate the first single‐base resolution map of 5hmC in rice ( Oryza sativa ), unveiling its stress‐responsive dynamics and regulatory interplay with 5mC during drought adaptation. Genome‐wide profiling revealed a basal 5hmC level of ~0.03 (defined as the ratio of C/(C + T) at each site), with drought triggering a pronounced reduction in 5hmC abundance and locus number, followed by incomplete recovery post‐rehydration. Unlike 5mC, which accumulates in heterochromatin, 5hmC preferentially localized to euchromatic regions, including promoters, exons, and intergenic elements, and exhibited enrichment at ABA‐responsive transcription factors (e.g., OsATAF1 , bZIP50 ). Strikingly, drought induced an antagonistic relationship between 5hmC and 5mC, with the latter increasing globally to reinforce transposon silencing. Multi‐omics analyses demonstrated that 5hmC depletion in promoters correlated with transcriptional downregulation, while its accumulation in gene bodies (notably 5′‐UTRs) suppressed stress‐responsive genes. These findings highlight 5hmC's bifunctional regulatory capacity, contingent on genomic context, and its role in balancing transcriptional plasticity with genome stability during stress. Our work establishes 5hmC as a dynamic epigenetic mark in plant environmental adaptation and provides a foundation for leveraging DNA hydroxymethylation in crop resilience engineering.