Epigenetic Control of Allele Transmission Reveals Hidden Stage-Specific Roles of Arabidopsis DNA Demethylases

Abstract Cytosine methylation is crucial for regulating gene expression, genomic imprinting, and silencing of transposable elements (TEs). Although heritable across cell divisions and generations, methylation is reversible and dynamically regulated throughout development. In Arabidopsis , active DNA demethylation is mediated by four DNA glycosylases—DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 (DML2), and DML3—via the base excision repair pathway. DME initiates demethylation in companion cells of the female and male gametes, leading to hypomethylation of maternal alleles in the endosperm. By contrast, ROS1, DML2, and DML3 (collectively RDD) primarily function during sporophytic growth to remove excess methylation. Due to the lack of distinct developmental phenotypes in rdd mutants when DME is present, the roles of RDD during reproduction, as well as their genetic interactions with DME in allele transmission, have remained largely unexplored. Here, we uncovered previously unrecognized functions of these demethylases through systematic genetic interaction analyses. We showed that rdd allele transmission is significantly impaired in a dme mutant background, suggesting that DME is required for proper RDD inheritance. Unexpectedly, dml3 mutation partially suppressed dme -mediated seed abortion in a gametophytic manner, suggesting a non-redundant and antagonistic interaction. Maternal ROS1 and DML2 are essential for the survival of rare dme homozygous seeds. Finally, rdd alleles displayed non-Mendelian segregation in a vegetative dme mutant background, implicating DME in vegetative epigenetic priming. These findings reveal stage-specific and interactive functions of Arabidopsis DNA demethylases, highlighting hidden reproductive roles for RDD and a previously unrecognized role for DME in the vegetative phase.