Abstract: DNA methylation is an essential epigenetic mark that 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 under-investigated on how the functional divergence of different DNMT duplicates have evolved among closely related species. Here, our study addressed evolutionary dynamic, transcriptional regulation and enzyme activities of all the 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 sub-functionalization (i.e., differential expression genes) is common mechanism that regulates 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) show complementary impacts on the transcriptional landscape. Our study provides evidence that, while DNA methylation of the three cytosine contexts (CG, CHG and CHH) are all 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 sub-functionalization, neo-functionalization, non-functionalization, Poaceae