Functional characterization of DEMETER-like DNA demethylases during growth-dormancy cycles in poplar

Resumen

ABSTRACT During their life cycle, plants are exposed to different environmental conditions that are often unfavorable. Their sessile condition has favored the acquisition of sophisticated molecular mechanisms that guarantee the plastic response to the different conditions faced during their development. Current advances indicate that changes in the functional state of chromatin by epigenetic modifications play important roles in the regulatory networks involved in plant responses to environmental cues. More specifically, several evidences in different plant species indicate that changes in the overall genomic DNA methylation promote tolerance to stressful environmental conditions, and also are associated with developmental programmed transition in response to the environment. The main aim of this doctoral thesis was to evaluate the involvement of DNA methylation in the control of the annual growth-dormancy cycle of trees, and the molecular characterization of hybrid poplar DEMETER like genes to evaluate their regulatory role in these transitions through modifying the DNA methylation profile. We performed an immunofluorescence-based protocol for the identification of epigenetic marks of closed and open chromatin states in hybrid poplar stems. The fluorescence signal of 5-methylcytosine was significantly higher in winter than in summer. Conversely, the fluorescence signal for acetylated Lys 8 of histone H4 was significantly higher in summer than in winter. Collectively, these results put forward an epigenetic control of winter dormancy in hybrid poplar stems. The DNA methylation pattern observed in the SAM, from dormancy to the time point of bud break, indicates that the reactivation of the cell cycle occurs after genomic DNA demethylation in hybrid poplar. In addition, we demonstrated that the active DNA demethylation carried out by DEMETER like 10 (PtaDML10) is required for bud break to allow the induction of cell metabolism genes needed for the reactivation of growth. Finally, we provide evidence that the chestnut DML (CsDML), the homolog of hybrid poplar PtaDML6, induces the bud formation needed for the survival of the apical meristem under the harsh conditions of winter, by inducing flavonoids biosynthesis accumulated in the SAM and bud scales during winter dormancy entrance.