The DNA methylation–demethylation balance prevents development of multiple megaspore mother cells in Arabidopsis

Female gametophyte development initiates from the megaspore mother cell (MMC), and only 1 somatic cell develops into an MMC. Here, we report that the balance between DNA methylation and demethylation ensures the maintenance of a single MMC in Arabidopsis (Arabidopsis thaliana). Surprisingly, a decrease or an increase of all 3 DNA methylation types (mCG, mCHG, and mCHH) caused abnormal enlargement of MMC-like cells and re-balancing DNA methylation rescued the enlarged MMC-like cells in the mutants of regulators for DNA methylation and demethylation. Systematic quantification of DNA methylation at the single-cell level demonstrated that mCHH levels begin to decrease from the central precursor MMC, preceding expression of the MMC marker KNUCKLES(KNU). Disrupting the regulation of DNA methylation caused the mCHH levels to become similar in the MMC and its neighboring cells, and these neighbors usually developed into MMC-like cells. Levels of the de novo DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) in the precursor MMC decreased before KNU expression, but the levels of the DNA glycosylase DEMETER decreased after KNU expression. Re-introduction of DRM2 or knockdown of DEMETER specifically in neighboring cells rescued the defects in drm1 drm2 double mutants. Collectively, our findings demonstrate that the balance of DNA methylation, rather than total methylation levels, facilitates maintenance of a single MMC.