Descripción: Under the current scenario of climate change, several Prunus spp. including sweet cherry (Prunus avium L.), have demonstrated special sensitivity when facing environmental challenges such as warmer winters, off-season frosts, and sudden spring rains. These events lead to serious damage in sweet cherry fruit production causing several disorders including altered cold accumulation. Cold accumulation during winter (dormancy) results fundamental for good quality flowering and, consequently, fruit productivity in sweet cherry. Two key and powerful cell mechanisms associated with gene expression control, namely small RNA (sRNA) pathways and epigenetic modulation, have arisen as competent and malleable pathways with a high involvement in the environmental sensing activity during cold accumulation events in dormancy. Small RNAs can guide cytosine methylation serving as an important layer of regulation in plants. These molecules are involved in modify chromatin and target gene expression via RNA interference (RNAi) pathways. During cold accumulation in tree flower buds, DNA methylation events and RNAi take place simultaneously co-localizing at different genome target zones or hot-spots; at the same time, a modulated synthesis of enzymes and proteins associated to methylation and demethylation processes scort the season. Small RNA biogenesis and DNA methylation machineries work together to reinforce different epigenetic conditions, leading to alternative states of gene activity that are not dictated by the linear DNA sequence. Recruitment of effector complexes that methylate DNA involves the association of small-RNA-guided protein complexes or RNA-binding proteins with nascent coding or non-coding RNA scaffolds. Both mechanisms act as re-inforcing loops and constitute the RNA-directed DNA methylation (RdDM). A second type of small RNA molecules, encoded by their ad-hoc genes and known as micro RNAs (miRNAs), are also able to generate direct mRNA cleavage, translational silencing, or transcriional silencing by DNA methylation after recognition of their target sequences. In previous works we showed that sRNAs/miRNAs and DNA methylation events (i.e. methylation of specific regulatory areas in dormancy associated genes) were associated to cold accumulation during winter and dormancy. These experiments showed that subsets of the total miRNA and small RNA population can be proposed as mediators of DNA methylation in relevant dormancy-associated genes. The present work will establish key small RNA molecules (both sRNAs/miRNAs), involved in DNA methylation during dormancy in sweet cherry. In a context of plant breeding, we thought that proposing these key small RNAs associated to RdDM during dormancy, their future biotechnological use can be possible. Through the use of an easy-to-design artificial miRNA (amiRNA), transient expression experiments in sweet cherry explants and stable genetic transformation of the model Arabidopsis, we propose the evaluation of RdDM potential of these candidate small RNAs obtained from sweet cherry's dormancy. Genetically modified sweet cherry explants and Arabidopsis plants with amiRNAs expressing small RNAs will be characterized to evaluate whether DNA methylation events taking place in sweet cherry can be replicated. The resulting molecules will be the first step for the development of new functional trait genomics and novel breeding innovation in Prunus spp. A better knowledge of the molecular bases of flower bud dormancy will strongly facilitate plant breeding tasks aimed at assessing the potential for environmental adaability of particular genotypes. Epigenetic modi?ers, as for instance our final small RNAs in this study, will become central to most molecular dormancy studies in the immediate future, with an increasingly important impact on development, environmental adaation, and biotechnology ?elds.
