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Vacuolar malic acid accumulation largely determines fruit acidity, a key trait for the taste and flavor of apple and other fleshy fruits. Aluminum‐activated malate transporter 9 (ALMT9/Ma1) underlies a major genetic locus, Ma, for fruit acidity in apple, but how the protein transports malate across the tonoplast is unclear. Here, it is shown that overexpression of the coding sequence of Ma1 (Ma1α) drastically decreases fruit acidity in "Royal Gala" apple, leading to uncovering alternative splicing underpins Ma1's function. Alternative splicing generates two isoforms: Ma1β is 68 amino acids shorter with much lower expression than the full‐length protein Ma1α. Ma1β does not transport malate itself but interacts with the functional Ma1α to form heterodimers, creating synergy with Ma1α for malate transport in a threshold manner (When Ma1β/Ma1α ≥ 1/8). Overexpression of Ma1α triggers feedback inhibition on the native Ma1 expression via transcription factor MYB73, decreasing the Ma1β level well below the threshold that leads to significant reductions in Ma1 function and malic acid accumulation in fruit. Overexpression of Ma1α and Ma1β or genomic Ma1 increases both isoforms proportionally and enhances fruit malic acid accumulation. These findings reveal an essential role of alternative splicing in ALMT9‐mediated malate transport underlying apple fruit acidity. [ABSTRACT FROM AUTHOR]

Background: Acidity is a major contributor to fruit quality. Several organic acids are present in apple fruit, but malic acid is predominant and determines fruit acidity. The trait is largely controlled by the Malic acid (Ma) locus, underpinning which Ma1 that putatively encodes a vacuolar aluminum-activated malate transporter1 (ALMT1)-like protein is a strong candidate gene. We hypothesize that fruit acidity is governed by a gene network in which Ma1 is key member. The goal of this study is to identify the gene network and the potential mechanisms through which the network operates. Results: Guided by Ma1, we analyzed the transcriptomes of mature fruit of contrasting acidity from six apple accessions of genotype Ma_ (MaMa or Mama) and four of mama using RNA-seq and identified 1301 fruit acidity associated genes, among which 18 were most significant acidity genes (MSAGs). Network inferring using weighted gene co-expression network analysis (WGCNA) revealed five co-expression gene network modules of significant (P < 0.001) correlation with malate. Of these, the Ma1 containing module (Turquoise) of 336 genes showed the highest correlation (0.79). We also identified 12 intramodular hub genes from each of the five modules and 18 enriched gene ontology (GO) terms and MapMan sub-bines, including two GO terms (GO:0015979 and GO:0009765) and two MapMap sub-bins (1.3.4 and 1.1.1.1) related to photosynthesis in module Turquoise. Using Lemon-Tree algorithms, we identified 12 regulator genes of probabilistic scores 35.5–81.0, including MDP0000525602 (a LLR receptor kinase), MDP0000319170 (an IQD2-like CaM binding protein) and MDP0000190273 (an EIN3-like transcription factor) of greater interest for being one of the 18 MSAGs or one of the 12 intramodular hub genes in Turquoise, and/or a regulator to the cluster containing Ma1. Conclusions: The most relevant finding of this study is the identification of the MSAGs, intramodular hub genes, enriched photosynthesis related processes, and regulator genes in a WGCNA module Turquoise that not only encompasses Ma1 but also shows the highest modular correlation with acidity. Overall, this study provides important insight into the Ma1-mediated gene network controlling acidity in mature apple fruit of diverse genetic background. [ABSTRACT FROM AUTHOR]