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.

Perennial deciduous trees such as Prunus avium undergo seasonal transitions, culminating in bud dormancy establishment that involves coordinated physiological and metabolic adjustments. Dormancy monitoring in orchard systems still relies primarily on temperature-based models and forcing assays, which rarely incorporate physiological or biochemical indicators. Here, we tested whether seasonal metabolic dynamics associated with dormancy progression differ between sweet cherry genotypes and whether these physiological differences are reflected in canopy-scale vegetation indices derived from satellite observations. Field measurements were conducted in two genotypes with contrasting chilling behavior (‘Regina’ and ‘210’) during the transition from vegetative growth to dormancy. Leaf gas exchange and chlorophyll fluorescence were monitored across the season, polar metabolites in floral buds were profiled by gas chromatography-mass spectrometry, and satellite-derived vegetation indices were used to characterize canopy dynamics. Dormancy progression was associated with declines in CO₂ assimilation, transpiration, PSII photochemical efficiency, and electron transport rate, accompanied by increases in intercellular CO₂ concentration and non-regulated energy dissipation. Metabolomic analysis revealed that genotype explained a larger proportion of metabolite variation than dormancy stage (PERMANOVA R² = 0.483, p = 0.001), while principal component analysis accounted for 79.7% of total variance. Fructose showed the strongest genotype difference during paradormancy I, corresponding to an approximately 9.5-fold increase in ‘Regina’. Pathway enrichment analysis highlighted starch and sucrose metabolism and pyruvate metabolism as the most represented pathways during dormancy progression. Satellite-derived vegetation indices captured seasonal canopy decline and were significantly associated with several physiological variables. These results provide an integrated description of physiological and metabolic adjustments during dormancy establishment in sweet cherry and highlight the potential of combining metabolomics, plant physiology, and open-access satellite observations to monitor phenological transitions in orchard systems at scalable spatial and temporal resolutions.