VcNADP-ME4 positively regulates malic acid degradation in blueberry fruit as modulated by two bZIPs

Fruit flavor in blueberry is highly determined by organic acid constitute and content. Previously, VcNADP-ME4 was preliminarily demonstrated to negatively involve in malic acid accumulation in rabbiteye blueberry fruits. To better understand the acid metabolism in this tree fruit, currently, the roles of VcNADP-ME4 in malic acid regulation was further unraveled. During fruit development of 'Tifblue', malic acid content exhibited an initial increase followed by a decrease, reaching its lowest level at the late developmental stage. In contrast, the expression of VcNADP-ME4 enhanced progressively and peaked at the ripening stage, indicating a negative correlation between VcNADP-ME4 expression and malic acid accumulation. Transient silencing of VcNADP-ME4 in the fruit significantly elevated malic acid levels, with stronger silencing effects leading to higher malic acid accumulation, whereas heterologous overexpression of this gene in tomato attenuated malic acid content. Subcellular localization analysis demonstrated that VcNADP-ME4 localized to the cell membrane. Further, it was revealed that the transcription factor VcbZIP44-like may bind to and activates the VcNADP-ME4 promoter, while VcGBF1 acts as a repressors to this gene. In conclusion, VcNADP-ME4 is a key gene regulating malic acid degradation in blueberry fruit, which offers new insights into the molecular mechanism underlying the organic acid metabolism of blueberry.