Loss of CG methylation inMarchantia Polymorphacauses disorganization of cell division and reveals unique DNA methylation regulatory mechanisms of non-CG methylation

DNA methylation is an epigenetic mark that ensures silencing of transposable elements (TEs) and affects gene expression in many organisms. The function of different DNA methylation regulatory pathways has been largely characterized in the model plant Arabidopsis thaliana . However, far less is known about DNA methylation regulation and functions in basal land plants. Here we focus on the liverwort Marchantia polymorpha , an emerging model species that represents a basal lineage of land plants. We identified Mp MET , the M. polymorpha orthologue of the METHYLTRANSFERASE 1 ( MET1 ) gene required for maintenance of methylation at CG sites in angiosperms. We generated Mp met mutants using the CRISPR/Cas9 system, which showed a significant loss of CG methylation and severe morphological changes and developmental defects. The mutants developed many adventitious shoot-like structures, suggesting that Mp MET is required for maintaining differentiated cellular identities in the gametophyte. Numerous TEs were up-regulated, even though non-CG methylation was highly increased at TEs in the Mp met mutants. Closer inspection of CHG methylation revealed features unique to M. polymorpha . Methylation of CCG sites in M. polymorpha does not depend on MET1 , unlike in A. thaliana and Physcomitrella patens . Furthermore, unlike A. thaliana , M. polymorpha shows higher methylation level at CAG sites than at other CHG contexts and CAG/CTG sites are mostly methylated asymmetrically. Interestingly, CAG and CTG methylation reached comparable levels and symmetry upon loss of CG methylation. Our results highlight the diversity of non-CG methylation regulatory mechanisms in plants.