Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)


Horticulture and Landscape Architecture

First Advisor

Jian-Kang Zhu

Committee Chair

Jian-Kang Zhu

Committee Member 1

Zhixiang Chen

Committee Member 2

Brian P. Dilkes

Committee Member 3

Kashchandra G. Raghothama


DNA methylation is an important epigenetic mark present in many eukaryotes, and is involved in many crucial biological processes, such as gene imprinting, regulation of gene expression, and genome stability. Proper genomic DNA methylation patterns are achieved through the concerted action of DNA methylation and demethylation pathways. In the model plant species Arabidopsis thaliana, ROS1 (REPRESSOR OF SILENCING 1) is one of the DNA demethylases and the key component in the demethylation pathway. Dysfunction of ROS1 leads to increase in DNA methylation level at thousands of genomic loci. However, the features of ROS1 targets are not well understood. In the first part of this dissertation, I will describe a study in which we identified and characterized genome-wide ROS1 target loci in Arabidopsis Col-0 and C24 ecotypes. In this study, we showed that ROS1 targets are associated with an enrichment of H3K18ac and H3K27me3, and with depletion of H3K27me1 and H3K9me2. Also we found that ROS1 can prevent the spreading of DNA methylation from highly methylated transposons to their nearby genes. Unexpectedly, we uncovered thousands of previously unidentified RdDM (RNA-directed DNA methylation) targets by analyzing the DNA methylome of ros1/nrpd1 double mutant plants. In addition, we showed that ROS1 also antagonizes RdDM-independent DNA methylation at more than one thousand genomic loci. Our results provide significant insights into the genome-wide effects of both ROS1-mediated active DNA demethylation and RNA-directed DNA methylation, as well as their interactions in plants. In the second part of this dissertation, I will describe another study in which we focused on AGO6 and AGO4, two Argonaute proteins involved in RdDM. AGO6 is generally considered to be redundant with AGO4 in RdDM. However, our genome-wide DNA methylation profiles and immunofluorescence localization analyses showed that redundancy between AGO4 and AGO6 is unexpectedly negligible in the genetic interactions and AGO4 and AGO6 mainly act sequentially in mediating RdDM.