6 mA methylation differences in Riccia fluitans organellar genomes under flood stress.

key messageThe first genome-wide evidence of 6 mA methylation in the organellar genomes of Riccia fluitans was provided by this study, with methylation patterns being shown to differ between aquatic and terrestrial forms, suggesting that epigenetic regulation is involved in environmental adaptation. DNA N⁶-methyladenine (6 mA) has recently emerged as an important epigenetic mark associated with transcriptional regulation and stress adaptation in plants. However, its role in organellar genomes remains poorly understood, especially in early-diverging lineages. The amphibious liverwort Riccia fluitans offers a unique opportunity to investigate environmentally induced epigenetic plasticity, as it can reversibly transition between terrestrial and aquatic morphotypes. Using Oxford Nanopore native DNA sequencing, we profiled 6 mA methylation across plastid and mitochondrial genomes of Riccia fluitans cultivated under aquatic and terrestrial conditions, and integrated these data with Illumina RNA-seq-based gene expression profiles. We identified 6 mA methylation sites characteristic of each habitat, with a markedly higher frequency of methylation in the aquatic form. Several differentially expressed genes (e.g., accD, psbA) were co-located with environment-specific methylation sites, suggesting potential spatial association between differential methylation and gene expression. Our results provide the first evidence of environmentally responsive organellar 6 mA methylation in a bryophyte, highlighting a previously unrecognized layer of epigenetic control that may contribute to the adaptive plasticity of early land plants.