Dynamics of DNA methylation and histone acetylation and methylation during European chestnut bud development

In perennial plants, tight regulation of bud development, from bud set, through dormancy, to bud burst, is critical for survival and is known to be regulated by epigenetic mechanisms. In Castanea sativa, a major temperate forest tree, the contribution of epigenetic regulators to bud development is poorly understood. To address this, we carried out an integrated study of bud morphology, immunodetection of epigenetic marks and gene expression. We analysed the distribution of two antagonistic epigenetic marks in the shoot apical meristem (SAM) by immunolocalizing 5-methylcytosine (5 mC) and histone H3 acetylated at Lysine 18 (H3K18ac), markers commonly associated with repressive and active chromatin states, respectively. Both marks displayed different distribution patterns in the SAM. In reproductive buds, distal SAM layers (L1, L2 and L3) showed increased 5 mC levels from bud set to dormancy, which then remained constant until burst, whereas H3K18ac levels were higher during bud set and dormancy but decreased at bud burst. To elucidate the influence of several chromatin modifiers in bud developmental stages, we profiled their expression throughout bud development. DNA chromomethylases (CsaCMT2 and CsaCMT3), the chromatin remodeler CsaSWC4 (SWR1 COMPLEX 4), the histone demethylase JUMONJI 21 (CsaJMJ21), CsaROS1 (REPRESSOR OF SILENCING 1) were highly expressed during active growth. The expression of all of these genes showed a significant positive correlation especially with maximum temperature, and several histone acetylases exhibited an opposite trend. Overall, this work indicates that small adjustments in DNA methylation levels, which are mediated by the coordinated expression of DNA methyltransferases and demethylases, alongside the dynamics of histone H3 acetylation, appear to play a role in the developmental processes of buds in C. sativa.