Primers used in this study.

Heterochromatin is critical for maintaining genome stability, especially in flowering plants, where it relies on a feedback loop involving the H3K9 methyltransferase, KRYPTONITE (KYP), and the DNA methyltransferase CHROMOMETHYLASE3 (CMT3). The H3K9 demethylase INCREASED IN BONSAI METHYLATION 1 (IBM1) counteracts the detrimental consequences of KYP-CMT3 activity in transcribed genes. IBM1 expression in Arabidopsis is uniquely regulated by methylation of the 7th intron, allowing it to monitor global H3K9me2 levels. We show the methylated intron is prevalent across flowering plants and its underlying sequence exhibits dynamic evolution. We also find extensive genetic and expression variations in KYP, CMT3, and IBM1 across flowering plants. We identify Arabidopsis accessions resembling weak ibm1 mutants and Brassicaceae species with reduced IBM1 expression or deletions. Evolution towards reduced IBM1 activity in some flowering plants could explain the frequent natural occurrence of diminished or lost CMT3 activity and loss of gene body DNA methylation, as cmt3 mutants in A. thaliana mitigate the deleterious effects of IBM1.