DNA methylation in plants is traditionally partitioned into CG, CHG and CHH contexts (with H any nucleotide but G), in an attempt to reflect mechanistic and functional differences between the pathways acting on these marks. By investigating DNA methylation patterns in trinucleotide contexts in four angiosperm species, we show that such a representation provides an incomplete picture, hiding large differences in motif use and mode of methylation in distinct chromosomal regions. CG methylation (mCG) is largely context-independent whereas, at CHG motifs, there is under-representation of mCCG in pericentric regions of A. thaliana and tomato and throughout the chromosomes of maize and rice. Pericentric regions also show large biases in CHH subcontext methylation, with CAA and CTA methylation far exceeding other contexts such as CTT in A. thaliana. By analysing a public collection of A. thaliana and maize mutants, as well as new gene-edited methylation mutants in tomato, we show that CCG hypomethylation relative to CAG/CTG methylation is driven by specificities of H3K9 methyltransferase SUVH family members. At CHH motifs, context-independent CHH methylation in A. thaliana and tomato is mediated by the RNA-directed DNA methylation process that is most active in the gene-rich euchromatin, whereas the contextual bias in heterochromatin is linked to the activity of chromomethyltransferases: CMT2 in A. thaliana and ZMET2 and ZMET5 in maize.

This analysis therefore reveals that the sequence context of the methylome of plant genomes is informative about the mechanisms of methylation and the overlying chromatin structure. Taking these effects into account will allow greater resolution in past and future analyses of methylomes,.