THE DNA METHYLATION LOCUS DDM1 IS REQUIRED FOR MAINTENANCE OF GENE SILENCING IN ARABIDOPSIS

To investigate the relationship between cytosine methylation and gene silencing in Arabidopsis, we constructed strains containing the ddm1 h ypomethylation mutation and a methylated and silenced PAI2 tryptophan biosynthetic gene (MePAI2) that results in a blue fluorescent plant ph enotype. The ddm1 mutation had both an immediate and a progressive eff ect on PAI gene silencing. Ln the first generation, homozygous ddm1 Me PAI2 plants displayed a weakly fluorescent phenotype, in contrast to t he strongly fluorescent phenotype of the DDM1 MePAI2 parent. After two generations of inbreeding by self-pollination, the ddm1/ddm1 lines be came nonfluorescent. The progressive loss of fluorescence correlated w ith a progressive loss of methylation from the PAI2 gene. These result s indicate that methylation is necessary for maintenance of PAI gene s ilencing and that intermediate levels of DNA methylation are associate d with intermediate gene silencing. The results also support our earli er hypothesis that ddm1 homozygotes act as ''epigenetic mutators'' by accumulating heritable changes in DNA methylation that can lead to cha nges in gene expression.