The callus formation capacity of strawberry leaf explant is modulated by DNA methylation.

Shoot regeneration from leaf tissue requires de-differentiation of cells from a highly differentiated state into an active dividing state, but how this physiological transition occurs and is regulated especially at epigenetic level remains obscure. Here we have characterized the DNA methylome represented by 5-methylcytosine (5mC) in leaf and the callus tissue derived from the leaf explant of woodland strawberry Fragaria vesca. We detected an overall increase of DNA methylation and distinct 5mC enrichment patterns in the CG, CHG and CHH sequence contexts in genetic and transposable elements. Our analyses revealed an intricate relation between DNA methylation and gene expression levels in leaf or leaf-derived callus. However, when considering the genes involved in callus formation and shoot regeneration, e.g. FvePLT3/7, FveWIND3, FveWIND4, FveLOG4 and FveIAA14, their dynamic transcription levels were associated with the differentially methylated regions located in the promoters or gene bodies, indicating a regulatory role of DNA methylation in the transcriptional regulation of pluripotency acquisition in strawberry. Furthermore, application of a DNA methyltransferase inhibitor 5'-azacytidine (5'-Aza) hampered both callus formation and shoot regeneration from the leaf explant. We further showed that 5'-Aza down-regulated the genes involved in cell wall integrity, such as expansin, pectin lyase and pectin methylesterase genes, suggesting an essential role of cell wall metabolism during callus formation. This study reveals the contribution of DNA methylation in callus formation capacity and will provide a basis for developing a strategy to improve shoot regeneration for basic and applied research applications.