Identification of AtALKBH1A and AtALKBH1D as DNA N6-adenine demethylases in Arabidopsis thaliana

DNA N6-methyladenine (6 mA) has recently been discovered as a novel DNA modification in animals and plants. In mammals, AlkB homolog 1 (ALKBH1) has been identified as a DNA 6 mA demethylase. ALKBH1 tightly controls the DNA 6 mA methylation level of mammalian genomes and plays important role in regulating gene expression. DNA 6 mA methylation has also been reported to exist in plant genomes, however, the plant DNA 6 mA demethylases and their function remain largely unknown. Here we identify homologs of ALKBH1 as DNA 6 mA demethylases in Arabidopsis. We discover that there are four homologs of ALKBH1, AtALKBH1A, AtALKBH1B, AtALKBH1C and AtALKBH1D, in Arabidopsis. In vitro enzymatic activity studies reveal that AtALKBH1A and 1D can efficiently erase DNA 6 mA methylation. Loss of function of AtALKBH1A and AtALKBH1D causes elevated DNA 6 mA methylation levels in vivo. atalkbh1a/1d mutant displays delayed seed gemination. Based on our RNA-seq data, we find some regulators of seed gemination are dysregulated in atalkbh1a/1d, and the dysregulation is correlated with changes of DNA 6 mA methylation levels. This study identifies plant DNA 6 mA demethylases and reports the function of DNA 6 mA methylation in regulating seed germination.