Differential regulation of heterochromatin and euchromatin by GmDDM1 during seed development ensures seedling viability in soybean

DECREASED IN DNA METHYLATION 1 (DDM1) is a chromatin remodeling factor that plays a major role in maintaining heterochromatin silencing and DNA methylation in plants. The soybean (Glycine max) genome, known for its large amount of pericentric heterochromatin, contains two DDM1 orthologs: GmDDM1a and GmDDM1b. In order to understand their functional roles in seed development, a process in which DNA methylation plays a significant role, we conducted mutant analysis. Single mutant and sesquimutant plants were healthy and produced seeds normally, but double mutants did not survive after germination. Methylome analysis of immature seeds detected an extensive decrease in methylation of CG and CHG sites in mutants. In fact, levels of CHG hypomethylation were correlated to the number of mutant alleles; however, severe CG methylation loss was only seen in double mutants. RNA-seq results showed that more than 3000 transposable elements (TEs) were activated transcriptionally in double mutants, which is consistent with the severe hypomethylation in their pericentromeric regions. Besides, we found 1388 protein-coding genes commonly increased in expression in all mutant genotypes ("Shared-up"), whereas 558 genes were only derepressed in double mutants ("Double-up"). These "Double-up" genes were largely found in heterochromatic regions, while "Shared-up" genes were mainly spread over the euchromatic chromosomal arms in which hypomethylation of mutants was substantially less than that of heterochromatin. These results explain the pivotal role of GmDDM1 in maintaining heterochromatin silencing and suggest the existence of a distinct GmDDM1-mediated euchromatin silencing mechanism during soybean seed development.