Vacuolar accumulation of malic acid, the predominant organic acid in apple (Malus domestica) and many other fleshy fruits, largely determines fruit acidity, a key trait for fruit taste and flavor. ALUMINUM-ACTIVATED MALATE TRANSPORTER 9 (ALMT9/Ma1) underlies a major genetic locus, Ma, for fruit acidity in apple. A natural mutation at base 1455 leads to a pre-mature stop codon that truncates the Ma1 protein by 84 amino acids to ma1, and this truncation significantly reduces Ma1’s malate transport activity by disrupting a conserved C-terminal domain, leading to low fruit acidity in recessive homozygous ma1ma1 genotypes. However, it remains unclear how Ma1 functions to transport malate across the tonoplast membrane. When the coding sequence of Ma1 was overexpressed in ‘Royal Gala’ apple (Ma1ma1) to determine its in planta function, to our surprise, the transgenic fruit had drastically reduced fruit acidity compared with the wild-type (WT). This led us to uncover that Ma1 undergoes alternative splicing. Two isoform proteins are generated by alternative splicing: Ma1beta being 68 amino acids shorter with much lower expression than the full-length protein Ma1alpha. Ma1beta does not transport malate itself, but interacts with the functional Ma1alpha, creating synergy with Ma1alpha for malate transport in a threshold manner when Ma1beta is equal to or exceeds 1/8 of Ma1alpha. In WT ‘Royal Gala’, Ma1 operates at this threshold. Overexpression of Ma1alpha triggers feedback inhibition on the native Ma1 expression via transcription factor MYB73, decreasing the Ma1beta level well below the threshold that leads to significant reductions in Ma1 function and malic acid accumulation in fruit. Overexpression of Ma1alpha and Ma1beta or genomic Ma1 increases both isoforms proportionally and enhances fruit malic acid accumulation. These findings reveal an essential role of alternative splicing in Ma1-mediated malate transport underpinning apple fruit acidity.
