show Abstracthide AbstractA contribution of DNA methylation to defense against invading nucleic acids and maintenance of genome integrity is uncontested; however, the relevance of this epigenetic mark for genome-wide gene regulation and plant development is unclear. Here, we knocked out all five known DNA methyltransferases in Arabidopsis, generating DNA methylation-free plants. This quintuple mutant exhibits a suite of unique and extreme developmental defects, unequivocally demonstrating that DNA methylation is essential for normal plant development. We show that CG methylation or non-CG methylation alone or jointly control multiple biological processes, including pavement cell shape, endoreduplication, cell death, flowering, trichome morphology, vasculature and meristem development, and root cell fate determination. Moreover, we found that DNA methylation has a strong dose-dependent effect in the regulation of gene expression and the repression of transposable element movement. Thus, our results demonstrate crucial roles of DNA methylation in orchestrating plant development and provide new insights into the function of this epigenetic mark in regulating gene expression in higher eukaryotes. Overall design: We performed strand-specific RNA sequencing, whole-genome bisulfite sequencing, and DNA sequencing