Artificial Sweet Cherry miRNA 396 Promotes Early Flowering in Vernalization-Dependent Arabidopsis Edi-0 Ecotype

The flowering and fruiting of sweet cherry (Prunus avium L.) depend on precise synchronization with seasonal events. During harsh autumn and winter conditions, floral buds enter dormancy to protect and prepare for the productive season. Dormancy release occurs after exposure to genotype-specific chilling temperatures, an event in which epigenetic reprogramming triggers further metabolic and gene expression activation. Similarly, several Arabidopsis ecotypes require chilling (vernalization) to transition from vegetative to floral states. At vernalization's end, the decrease in the repressor complex formed by SHORT VEGETATIVE PHASE (SVP) and FLOWERING LOCUS C (FLC) allows FLOWERING LOCUS T (FT) to induce flowering. However, this alone does not fully explain the process. MicroRNAs (miRNAs) play a crucial role in gene regulation during plant development and environmental interactions, and miR396's role during flower development and vernalization has been described in some plant species, although not for sweet cherry dormancy. We used 'Regina', a high-chill sweet cherry variety, to identify candidate small RNA molecules throughout dormancy, resulting in the detection of miR396. The transcript expression levels of the putative miRNA target genes were evaluated through quantitative PCR analyses of dormant buds. Additionally, an artificial sweet cherry miR396 was used to transform Arabidopsis Edi-0, a vernalization-requiring ecotype. Ectopic expression of this artificial molecule partially mirrored the effect on target genes observed in dormant buds and, more importantly, led to vernalization-independent flowering. Artificial miR396 expression also resulted in decreased FLC and increased SVP and FT transcript levels. These results could pave the way for future studies on the involvement of miR396 in the regulation of dormancy and flowering, with potential applications in improving crop resilience and productivity.