Characterization and fine mapping of Arabidopsis RLL3, a locus required for DNA demethylation pathway

DNA methylation pattern is found to be established by the combined actions of DNA methylation and demethylation. Compared to the DNA methylation pathway, DNA demethylation pathway, however, remains largely unknown. To better understand the DNA demethylation pathway, we performed genetic screening for Arabidopsis mutants with increased genomic DNA methylation levels through a 2 × 35S:LUC (LUC, luciferase) reporter system. A mutant with reduced LUC luminescence was identified by such a system, therefore named rll3-1 (for reduced LUC luminescence 3-1). The rll3-1 mutant exhibited pleiotropic developmental defects, such as delayed bolting as well as flowering, more branches, etc. By map-based cloning approach, rll3 locus that contains a single nuclear recessive mutation as revealed by the genetic analysis was mapped to a region between molecular markers CL102_B1 M1 and CL102_B3M1, which are located in bacterial artificial chromosome (BAC) clones F9P14 and F12K11, respectively, on chromosome 1. Chop-PCR analysis indicated that a total of seven tested loci displayed elevated DNA methylation levels. Whole-genome bisulfite sequencing further revealed 1536 loci exhibiting increased DNA methylation levels relative to Col-LUC control, among which there are 507 such loci overlapping between the rll3-1 and ros1-7 mutants, suggestive of a functional association between RLL3 and REPRESSOR OF SILENCING 1 (ROS1). Further investigations demonstrated that the expression levels of a few genes (like ROS1, IDM1, etc.), which are involved in DNA demethylation pathway, remained unchanged in the rll3-1 mutant, indicating that the increased DNA methylation levels in rll3-1 mutant are not attributable to downregulation of such genes. Taken together, our studies provide a demonstration of the involvement of RLL3 in the DNA demethylation pathway.