Genetic Screening Of Arabidopsis Mutants Associated With DNA Demethylation Processes And Mapping As Well As Functional Analysis Of DNA Demethylation Genes

To unravel the molecular mechanisms of DNA demethylation processes in Arabidopsis,we used two ways to discover genes involved in such processes in this study.We then investigated the functions of these genes,and explored their roles in contributing to the DNA demethylation.Scheme A:We isolated a low-luminescence mutant referred to as rll2(reduced LUC luminescence 2)from an ethyl methanesulfonate(EMS)-mutagenized M2 population derived from a Col-LUC transgenic line that carries a 2X35S:LUC(LUC:luciferase)reporter as well as emits high LUC luminescence in normal growth conditions.The wild-type gene defined by the mutation was hence termed RLL2.Compared to the Col-LUC,however,the rll2 mutant emitted quite low luminescence,and exhibited obvious dwarfism phenotype.Genetic analyses revealed that rll2 bears a single nuclear-encoded recessive mutation that is responsible for the low LUC luminescence phenotype.We further mapped the RLL2 gene on chromosome 5 and then narrowed down the rll2 mutation to a small region between the markers CL506-B14M1 and CL507-B6M1 which are located at BAC clones F5024 and T6G21,respectively.Chop-PCR results demonstrated that several genomic loci are hypermethylated in rll2,and RT-PCR data indicated that the expression of a few endogenous genes targeted by RNA-directed DNA methylation(RdDM)pathway decreased to varying extents in such a mutant.Taken together,these results suggest that RLL2 might be involved in DNA demethylation processes.Scheme B:By using gene co-expression analysis,we obtained a gene named IDP1(Increase DNA methylation Protein 1),which was co-expressed with ROSl.We identified a couple of T-DNA insertional homozygous idpl mutants by genotyping and RT-PCR methods,and confirmed that they are all loss-of-function alleles.Chop-PCR results demonstrated that several genomic loci are hypermethylated in idpl.Complementation of idpl mutant with IDP1 gDNA decreased its hypermethylation to varying levels,some of which are close to methylation levels in wild type control.GUS staining assays revealed that IDP1 is mainly expressed in cotyledons and sepals,and GUS staining was predominantly detected in veins.By analyzing the DNA methylation levels of four double mutants,i.e.idpl-1/rosl-4,idpl-1/nrpd1a-3,idpl-1/rdml-4 and idp1-1/met1(Col-gl),we verified that IDP1 indeed participates in the processes of DNA demethylation,and it was antagonistic to the effects of RdDM pathway.The performance of the LUC luminescence and Kanamycin resistance of two genotypes idpl-1/Col-LUC and idp1-1/C24-LUC(Col-4)demonstrated that IDP1 is partially required for activation of the 35S promoter.Genome-wide bisulfite sequencing analysis showed that there are some similarities and differences between target regions of IDP1 and ROS1.Taken together,as a regulator in DNA demethylation processes,IDP1 may operate in the same pathway with ROS1,but its target regions on genomic DNA do not completely overlap with those of ROS1.