Regulation of Active DNA Demethylation and its Role in Fertility in Arabidopsis thaliana

Abstract

The genome of Arabidopsis thaliana must maintain control over the balance between de novo methylation, maintenance of existing methylation, and active demethylation as it utilizes these processes to facilitate gene expression changes throughout the lifetime of the plant. Expression of the DNA demethylase REPRESSOR OF SILENCING1 (ROS1) is dependent upon this balance as well as perpetuating it, yet many aspects of the regulation of ROS1 remain unknown. In this work, I show that the downregulation of ROS1 in mutants of the RNA-directed DNA methylation pathway occurs at the transcriptional level, and is dependent upon an 817-bp region in the proximal promoter region of ROS1. The deletion of this region using CRISPR-Cas9 technology resulted in increased expression of ROS1 in both wildtype and methylation-deficient backgrounds, indicating that this region may be a methylation-sensitive silencer sequence. Additional deletions in the endogenous chromosome identified further regions that contain regulatory elements of ROS1. Additionally, I further investigated the results when the balance between methylation and active demethylation is disturbed, by characterizing a quadruple mutant of all four member of the DEMETER family of DNA glycosylases in somatic tissues: dme;ros1;dml2;dml3 (drdd). This mutant displays an early flowering phenotype which was linked to downregulation of the floral repressor FLOWERING LOCUS C, concurrent with DRDD-dependent hypermethylation in the 5’ flanking region. I also characterized a low-penetrance male fertility defect in drdd mutants, which I determined is caused by a delay in anther dehiscence that could be a result of altered reactive oxygen species accumulation. This work has led to an increase in our understanding of the mechanisms by which ROS1 is regulated, and the mechanisms by which active demethylation affect transcription and development of the plant.