Determination of DNA methylation and histone modification kinetic in regulatory regions of Dormancy Associated MADS-box gene (DAM6) in sweet cherry (Prunus avium) during bud dormancy release

Bud dormancy release in Rosaceae depends on a minimum accumulation of chilling hours. DORMANCY ASSOCIATED MADS-BOX (DAM) genes are negative flowering regulators and strong candidates for the regulation of bud growth cessation and dormancy maintenance. Epigenetic mechanisms appear to control the silencing of DAM genes in peach in a similar way to its ortologous in Arabidopsis during vernalization. To better understand the molecular mechanism of dormancy release in sweet cherry (Prunus avium var. Bing); we isolated the promoter and the first intron of DAM6. Histone modifications and DNA methylation pattern kinetic of these regions, in response to chilling hours (CH) accumulation, was studied in floral buds sampled weekly during the dormancy period. Using Chromatin Immunoprecipitation (ChIP) essay, we observed that the presence of H3K27me3 and histone variant H2A.Z increased reaching a maximum at 1038 CH in the promoter. This histone modification and histone variant are related to silencing of gene expression. On the other hand it was observed that the presence of H3K4me3, a modification related to gene activation, decreases with CH accumulation in the promoter. In parallel, the results obtained from bisulfite sequencing show an increase of DNA methylation in the first intron of DAM6, and suggest a dynamic change of methylated cytosines, as the chilling requirement is being fulfilled indicating silencing of DAM6. Our results strength the role of epigenetic mechanisms in chilling requirement for dormancy release and blooming in Rosaceous species as sweet cherry. This work is supported by FONDEF G09I1008; FONDAP CRG 15070009 and Basal PFB-16.