Leaf senescence is driven by the expression of senescence-associated genes (SAGs).Development-specific genes often undergo DNA demethylation in their promoter and other regions,which regulates gene expression.Whether and how DNA demethylation regulates the expression of SAGs and thus leaf senescence remain elusive.Whole-genome bisulfite sequencing (WGBS) analyses of wild-type (WT) and demeter-like 3 (dml3) Arabidopsis leaves at three developmental stages revealed hypermethylation during leaf senescence in dml3 compared with WT,and 20 556 differentially methylated regions (DMRs) were identified by comparing the methylomes of dml3 and WT in the CG,CHG,and CHH contexts.Furthermore,we identified that 335 DMR-associated genes (DMGs),such as NAC016 and SEN1,are upregulated during leaf senescence,and found an inverse correlation between the DNA methylation levels (especially in the promoter regions) and the transcript abundances of the related SAGs in WT.In contrast,in dml3 the promoters of SAGs were hypermethylated and their transcript levels were remarkably reduced,and leaf senescence was significantly delayed.Collectively,our study unraveled a novel epigenetic regulatory mechanism underlying leaf senescence in which DML3 is expressed at the onset of and during senescence to demethylate promoter,gene body or 3’UTR regions to activate a set of SAGs.