The single-berry metabolomic clock paradigm reveals new stages and metabolic switches during grapevine berry development

Asynchronicity causes metabolic chimerism in currently used scales of grapevine phenology, calling for a re-examination of berry development at the individual fruit scale. To reveal the dynamics of metabolite composition in grapevine berries without a priori phenological assumptions, a dataset of 9256 ions was obtained for 125 fruits at different stages by non-targeted ultra-performance liquid chromatography coupled to high-resolution mass spectrometry. This large metabolomic dataset was submitted to an analysis workflow combining classification and dimension reduction tools. Our analysis resulted in the clustering of metabolites into 12 specific kinetic patterns and a metabolome-based definition of the pericarp intrinsic clock that outperformed a timing scheme based on more traditional methods of photographic observations and quantification of sugars and organic acids. This increased temporal resolution enabled the identification of metabolite clusters that are indicative of the onset of ripening, notably characterized by transient lipidic changes and the start of abscisic acid accumulation. A cluster of stilbenes that accumulated during terminal fruit shriveling after the arrest of phloem unloading was also highlighted. Our non-targeted approach thus enabled a more precise characterization of grapevine berry development through the concept of the metabolomic clock. The discovery of new metabolic milestones of berry development paves the way toward a better assessment of the impact of environmental changes on the metabolism of non-climacteric fruit in different genotypes.Untargeted metabolomics coupled with single-fruit sampling throughout the whole developmental cycle of grapevine berries enables the fine characterization of fruit phenology and the identification of metabolite clusters specific to certain key stages.