An Investigation into the Evolution and Function of RNA-Directed DNA Methylation and its Component Proteins

Abstract

RNA-directed DNA methylation (RdDM) is a major epigenetic pathway in plants that is required to establish methylation in all contexts and maintain CHH methylation. The function of RdDM is mediated by multiple proteins that are still being characterized in land plants. Pol IV and Pol V form a major part of the RdDM process, and the subunits that compose these polymerases have evolved independently across the land plant lineage, resembling a stepwise manner of evolution. While a Pol IV/Pol V-like first subunit exists in some species in the Charophytic Green Algae (CGA) – the closest relatives of land plants, not much is known about the identity of the protein, and the extent to which this ancestral protein can be identified in the different CGA lineages. Ongoing duplications of Pol IV and Pol V subunits have been reported in different lineages of land plants. One such example is specific to Poaceae, where duplication and diversification of Pol V subunits have led to evolution of a putative sixth polymerase. The expansion of the RdDM pathway in land plants is very intriguing, because RdDM is important in plant development. Multiple plant species show high levels of methylation in the embryo, and there is no clear understanding about if plant embryos gain that methylation autonomously or if surrounding tissues in a seed play a role.Thus, major questions remain in the field about how RNA polymerases IV and V have evolved, particularly in regard to their core catalytic subunits. Questions also remain about the function of the putative sixth polymerase in grasses and how mature embryos in different species gain high amounts of DNA methylation. This study attempts to understand how RNA polymerases IV and V have evolved, by looking across the green plant lineage, particularly focusing on CGAs. I report the presence of a Pol IV/Pol V associated protein of the chromatin remodeler family in the CGAs. Our results show that homologs of Pol IV and Pol V first and second subunit evolved around the same time point as the Pol IV/Pol V-associated chromatin remodeler homolog that is identifiable in the CGAs, and thereby provides a more nuanced insight into the evolution of RdDM components. From our analysis into a T-DNA mutation in the second subunit of Pol IV and Pol V, it can be hypothesized that this subunit works non-redundantly in leaf tissues Pol VI, a putative novel polymerase unique to grasses, and to explore the molecular function of Pol VI, I have targeted these subunit genes with CRISPR-Cas9. My exploration into the methylation status of mature embryos in Brassica rapa, where loss of RdDM causes severe developmental defects, shows the hypermethylated nature of those embryos. Analyzing small RNA and methylation data across different tissues in Brassica rapa, I have shown that the methylation in the embryo is autonomous.