Somatic DNA demethylation generates tissue-specific methylation states and impacts flowering time [RNA-seq]

Cytosine methylation is a reversible epigenetic modification to DNA. In plants, removal of cytosine methylation is accomplished by the four members of the DME family of 5-methylcytosine DNA glycosylases. Demethylation by DME is critical for seed development. Consequently, determining the function of the entire gene family in somatic tissues by mutant analysis has not been possible. Here, we bypassed the reproductive defects of dme mutants to create somatic quadruple homozygous mutants of the entire DME family. dme; ros1; dml2; dml3 (drdd) leaves exhibit hypermethylated genomes compared to both wild-type plants and rdd triple mutants, indicating functional redundancy among all four demethylases. Targets of demethylation include regions co-targeted by RNA-directed DNA methylation and, surprisingly, CG gene body methylation, indicating dynamic methylation at these less-understood sites. Additionally, many tissue-specific methylation differences are absent in drdd, suggesting a role for active demethylation in generating divergent epigenetic states across wild-type tissues. Furthermore, drdd plants display an early flowering phenotype, which is correlated with 5’ hypermethylation and transcriptional down-regulation of FLOWERING LOCUS C. Active DNA demethylation is therefore required for proper methylation patterning across somatic tissues and defines the epigenetic landscape of both intergenic and coding regions. Whole genome bisulfite sequencing (BS-seq), enzymatic-methyl (EM) sequencing and RNA-seq of Arabidopsis thalana DNA demethylation mutants