Heat-induced CHH methylation changes near an F-box protein gene in Arabidopsis mature-stage seeds

Plants respond to heat stress through dynamic changes in DNA methylation, particularly in the CHH context. However, whether these changes occur in specific gene classes and vary across developmental stages remains poorly understood. Here, I re-analyzed publicly available whole-genome bisulfite sequencing data from Arabidopsis bent-stage embryos and mature-stage seeds exposed to 27 °C (moderate heat exposure) and 23 °C (control). I found that heat-induced CHH methylation targets specific genomic loci in a developmental stage-specific manner, with a differentially methylated window located within 6 kb of AT5G44410, an F-box protein-encoding gene. AT5G44410 showed increased transcript abundance under heat stress (56.4 → 155.5 transcripts per million; descriptive 2.8-fold change), suggesting a possible role for protein degradation in heat acclimation. Using replicate-consistent analysis (requiring differentially methylated windows to be present in both biological replicates), I found that 85–88% of single-replicate differentially methylated windows were not reproducible across biological replicates under the applied threshold, highlighting the necessity of biological replication in whole-genome bisulfite sequencing studies. Among a focused subset of specialized metabolism genes (n = 75), no differentially methylated windows were detected in the promoter regions. These findings demonstrate that heat-induced CHH methylation occurs at specific genomic loci in a developmental stage-specific manner and identify AT5G44410 as a candidate F-box gene associated with increased transcript abundance under heat stress. Functional validation is required to establish causal relationships. Replicate-consistent analysis revealed stage-specific CHH methylation responses to heat stress and identified AT5G44410 as a candidate heat-responsive F-box gene.