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

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.