Global hypermethylation of the N(6)-methyladenosine RNA modification associated with apple heterografting

Grafting can facilitate better scion performance and is widely used in plants. Numerous studies have studied the involvement of mRNAs, small RNAs, and epigenetic regulations in the grafting process. However, it remains unclear whether the mRNA N(6)-methyladenosine (m(6)A) modification participates in the apple (Malus x domestica Borkh.) grafting process. Here, we decoded the landscape of m(6)A modification profiles in ‘Golden delicious’ (a cultivar, Gd) and Malus prunifolia ‘Fupingqiuzi’ (a unique rootstock with resistance to environmental stresses, Mp), as well as their heterografted and self-grafted plants. Interestingly, global hypermethylation of m(6)A occurred in both heterografted scion and rootstock compared with their self-grafting controls. Gene Ontology (GO) term enrichment analysis showed that grafting-induced differentially m(6)A-modified genes were mainly involved in RNA processing, epigenetic regulation, stress response, and development. Differentially m(6)A-modified genes harboring expression alterations were mainly involved in various stress responses and fatty acid metabolism. Furthermore, grafting-induced mobile mRNAs with m(6)A and gene expression alterations mainly participated in ABA synthesis and transport (e.g. carotenoid cleavage dioxygenase 1 [CCD1] and ATP-binding cassette G22 [ABCG22]) and abiotic and biotic stress responses, which might contribute to the better performance of heterografted plants. Additionally, the DNA methylome analysis also demonstrated the DNA methylation alterations during grafting. Downregulated expression of m(6)A methyltransferase gene MdMTA (ortholog of METTL3) in apples induced the global m(6)A hypomethylation and distinctly activated the expression level of DNA demethylase gene MdROS1 (REPRESSOR OF SILENCING 1) showing the possible association between m(6)A and 5mC methylation in apples. Our results reveal the m(6)A modification profiles in the apple grafting process and enhance our understanding of the m(6)A regulatory mechanism in plant biological processes.