Abstract: DNA methylation is an essential epigenetic mark that is involved in a range of biological activities in all domains of life. Molecular mechanisms underlying how the DNA methyltransferases (DNMTs) catalyze cytosine methylation have been well documented in model species. However, it still remains underinvestigated as to how the functional divergence of different DNMT duplicates has evolved among closely related species. Here, our study addressed evolutionary dynamic, transcriptional regulation and enzyme activities of all three DNMTs (DNMT1, DNMT2, and DNMT3) in extant Poaceae species. Our results show that, although all Poaceae species are derived from the most recent common ancestor, biased genetic fractionation acting on different DNMT duplicates has resulted in high copy number variations among extant species. In addition, expression-level subfunctionalization (i.e., differential expression genes) is a common mechanism that regulates the transcriptional pattern of different DNMT duplicates in extant Poaceae species. Neo-functionalization and positive selection further promote functional divergence (i.e., different catalytic efficiency) among different DNMT duplicates. In particular, estimates of enzyme activities demonstrate that highly expressed gene copies of the DNMT1 (i.e., MET1a and MET1b) tend to show high catalytic efficiency. Furthermore, functional analyses of seven DNMT mutants also reveal that loss of function of three DNMT genes (OsCMT3a, OsCMT2, and OsDRM2) exerts complementary impacts on the transcriptional landscape. Our study provides evidence that, while DNA methylation of all three cytosine contexts (CG, CHG, and CHH) is catalyzed by the three DNMTs, different mechanisms have together promoted high evolutionary dynamic and functional divergence in extant Poaceae species.

Key words: cytosine methylation, expression-level subfunctionalization, neo-functionalization, non-functionalization, Poaceae