Metabolite Profiling Reveals Developmental Inequalities in Pinot Noir Berry Tissues Late in Ripening

Uneven ripening in Vitis vinifera is increasingly recognized as a phenomenon of interest, with substantial implications for fruit and wine composition and quality. This study sought to determine whether variation late in ripening (∼Modified Eichhorn-Lorenz stage 39) was associated with developmental differences that were observable as fruits within a cluster initiated ripening (véraison). Four developmentally distinct ripening classes of berries were tagged at cluster véraison, sampled at three times late in ripening, and subjected to untargeted HPLC-MS to measure variation in amino acids, sugars, organic acids, and phenolic metabolites in skin, pulp, and seed tissues separately. Variability was described using predominantly two strategies. In the first, multivariate analysis (Orthogonal Projections to Latent Structures-Discriminant Analysis, OPLS-DA) was used to determine whether fruits were still distinguishable per their developmental position at véraison and to identify which metabolites accounted for these distinctions. The same technique was used to assess changes in each tissue over time. In a second strategy and for each annotated metabolite, the variance across the ripening classes at each time point was measured to show whether intra-cluster variance (ICV) was growing, shrinking, or constant over the period observed. Indeed, berries could be segregated by OPLS-DA late in ripening based on their developmental position at véraison, though the four ripening classes were aggregated into two larger ripening groups. Further, not all tissues were dynamic over the period examined. Although pulp tissues could be segregated by time sampled, this was not true for seed and only moderately so for skin. Ripening group differences in seed and skin, rather than the time fruit was sampled, were better able to define berries. Metabolites also experienced significant reductions in ICV between single pairs of time points, but never across the entire experiment. Metabolites often exhibited a combination of ICV expansion, contraction and persistence. Finally, we observed significant differences in the abundance of some metabolites between ripening classes that suggest the berries that initiated ripening first remained developmentally ahead of the lagging fruit even late in the ripening phase. This presents a challenge to producers who would seek to harvest at uniformity or at a predefined level of variation.