The aluminum-activated malate transporter 9 (VaALMT9) promotes malate accumulation in rabbiteye blueberry fruit and is transcriptionally inhibited by VaMYB1R1 and VaWRKY21.

Blueberry (Vaccinium spp.) has gained worldwide popularity as a fruit crop of exceptional flavor and high nutritional value. Fruit acidity, a critical determinant of consumer preference in rabbiteye blueberry, is primarily governed by vacuolar malic acid accumulation; however, the underlying regulatory mechanism remains unclear. In the current study, VaALMT9, encoding an aluminum-activated malate transporter, was identified from rabbiteye blueberry transcriptome data. The full-length coding sequence encodes a 574 amino acid protein harboring the characteristic WEP fingerprint motif of the ALMT family. Subcellular localization analysis suggested that VaALMT9 localizes to the endomembrane system, most likely the tonoplast (vacuolar membrane). Expression analysis revealed that VaALMT9 transcript levels paralleled malate accumulation during fruit development, with both peaking at the purple stage and declining at the late ripening stage. Virus-induced gene silencing of VaALMT9 in blueberry fruits significantly reduced its transcript levels (by 96.74%) and malate contents of the two independent silenced lines were remarkably decreased by 0.62 mg/g and 0.50 mg/g, which were reduced by 19.37% and 15.78% in comparison with those of the control. Conversely, VaALMT9-overexpressing tomato markedly increased malate accumulation by 13.79-44.37%. Moreover, yeast one-hybrid and dual-luciferase reporter assays revealed that VaMYB1R1 and VaWRKY21 bind to the VaALMT9 promoter and repress its transcriptional activity. In conclusion, VaALMT9 was identified as a key gene promoting malate accumulation, and is transcriptionally inhibited by VaMYB1R1 and VaWRKY21. The findings provide new insights into the molecular regulation underlying fruit acid accumulation and offer valuable gene targets for genetic improvement of fruit quality in this fruit crop.