Sustained carbon import supports sugar accumulation and anthocyanin biosynthesis during fruit development and ripening in blueberry (Vaccinium ashei).

Fruit ripening is a highly coordinated process involving molecular and biochemical changes that collectively determine fruit quality. The underlying metabolic programs and their transitions leading to fruit ripening remain largely under-characterized in blueberry (Vaccinium sp.), which exhibits atypical climacteric behavior. In this study, we focused on sugar, acid and anthocyanin metabolism in two rabbiteye blueberry cultivars, Premier and Powderblue, during fruit development and ripening. Concentrations of the three major sugars, sucrose (Suc), glucose (Glc), and fructose (Fru) increased steadily during fruit development leading up to ripening, and increased dramatically by around 2-fold in 'Premier' and 2- to 3-fold in 'Powderblue' during the final stage of fruit ripening. Starch concentration was very low throughout fruit development in both cultivars indicating that it does not serve the role of a major transitory carbon (C) storage form in blueberry fruit. Together, these patterns indicate continued import of C, likely in the form of Suc, throughout blueberry fruit development. Concentrations of the predominant acids, malate and quinate, decreased during ripening, and may contribute to increased shikimate biosynthesis which, in-turn, allows for downstream phenylpropanoid metabolism leading to anthocyanin synthesis. Consistently, anthocyanin concentrations were highest in fully ripened blue fruit. Weighted gene co-expression network analysis (WGCNA) was performed using a 'Powderblue' fruit ripening transcriptome and targeted fruit metabolite concentration data. A 'dark turquoise' module positively correlated with sugars and anthocyanins, and negatively correlated with acids (malate, quinate), was identified. Gene Ontology (GO) enrichment analysis of this module identified transcripts related to sugar, acid, and phenylpropanoid metabolism pathways. Among these, increased transcript abundance of a VACUOLAR INVERTASE during ripening was consistent with sugar storage in the vacuole. In general, transcript abundance of the glycolysis pathway genes was upregulated during ripening. The transcript abundance of PHOSPHOENOLPYRUVATE (PEP) CARBOXYKINASE increased during fruit ripening and was negatively correlated with malate concentration, suggesting increased malate conversion to PEP, which supports anthocyanin production during fruit ripening. This was further supported by the co-upregulation of several anthocyanin biosynthesis-related genes. Together, this study provides insights into important metabolic programs, and their underlying gene expression patterns during fruit development and ripening in blueberry.