Embryo-specific epigenetic mechanisms reconstitute the CHH methylation landscape during Arabidopsis embryogenesis

The modification of flowering plant DNA by CHH methylation acts primarily to silence transposable elements, of which many active copies are present in Arabidopsis thaliana. During embryogenesis, the CHH methylation landscape is dramatically reprogrammed, resulting in exceedingly high levels of this modification upon mature embryo formation. The mechanisms constituting the remodeling process, and its function in embryos, are unclear. Here, we isolate embryos from Arabidopsis plants harboring mutations for key components of the pathways that confer CHH methylation, namely RNA-directed DNA methylation (RdDM) and the Chromomethylase 2 (CMT2) pathways. We reveal that embryos are more methylated than leaves at shared CMT2 and RdDM targeting loci, accounting for most embryonic CHH hypermethylation. While the majority of embryo CHH methylated loci overlap with those in somatic tissues, a subset of conventional pericentric CMT2-methylated loci are instead targeted by RdDM in embryos. These loci, termed embRdDM exhibit intermediate H3K9me2 levels, associated with increased chromatin accessibility. Strikingly, more than 50% of the embRdDM loci in pollen vegetative (nurse) cells and ddm1 mutant somatic tissues are also targeted by RdDM, and these tissues were also reported to exhibit increased chromatin accessibility in pericentric heterochromatin. Furthermore, the root columella stem cell niche also displays CHH hypermethylation and an enriched presence of small RNAs at embRdDM loci. Finally, we observe a significant overlap of CHH hypermethylated loci with endosperm DEMETER targeting sites, suggesting that non-cell autonomous communication within the seed may contribute to the epigenetic landscape of the embryo. However, similar overlap with vegetative cell DEMETER targets indicates that the chromatin landscape that allows DEMETER access is mirrored in developing embryos, permitting CHH methylation catalysis at the same loci. Our findings demonstrate that both conserved and embryo-specific epigenetic mechanisms reshape CHH methylation profiles in the dynamic chromatin environment of embryogenesis.