Temperate fruit trees belonging to Prunus species have the ability to suspend (induce dormancy) and resume growth periodically in response to environmental and seasonal conditions. Endodormancy release requires the long-term accumulation of chill. Upon accumulation of cultivar-specific chill requirements, plants enter the state of ecodormancy, which means the ability to grow has been restored, depending on the fulfilment of heat requirements. As many different metabolic pathways are implicated in endodormancy release, we have performed a metabolomic analysis, using the ultra-high-performance liquid chromatography–quadrupole time-of-flying (UPLC–QToF) technique. We assayed flower buds in different stages of endodormancy in four almond cultivars with different flowering times: the extra-early Desmayo Largueta, the late Antoñeta, the extra-late Penta, and the ultra-late Tardona. An orthogonal projection to latent-structure discriminant-analysis model was created to observe differences between endodormant and ecodormant flower buds. The metabolites showing the most significant variation were searched against the Metlin, HMDB, and KEGG libraries, which allowed us to identify 87 metabolites. These metabolites were subsequently assigned to specific pathways, such as abscisic acid biosynthesis, phenylpropanoid biosynthesis, and D-sorbitol metabolism, among others. The two metabolites that exhibited the most significant variations in all the cultivars studied with fold changes of up to 6.49 were ascorbic acid and prunasin. For the first time, these two metabolites have been proposed as potential biomarkers for endodormancy release in almond. Given the high synteny present between the Rosaceae species, these results could be extrapolated to other important crops like peach, plum, cherry, or apricot, among others.

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