Apple ALMT9 Requires a Conserved C-Terminal Domain for Malate Transport Underlying Fruit Acidity

Abstract

Malate accumulation in the vacuole largely determines apple (<i>Malus domestica</i>) fruit acidity, and low fruit acidity is strongly associated with truncation of <i>Ma1</i>, an ortholog of <i>ALUMINUM-ACTIVATED MALATE TRANSPORTER9</i> (<i>ALMT9</i>) in Arabidopsis (<i>Arabidopsis thaliana</i>). A mutation at base 1,455 in the open reading frame of <i>Ma1</i> leads to a premature stop codon that truncates the protein by 84 amino acids at its C-terminal end. Here, we report that both the full-length protein, Ma1, and its naturally occurring truncated protein, ma1, localize to the tonoplast; when expressed in <i>Xenopus laevis</i> oocytes and <i>Nicotiana benthamiana</i> cells, Ma1 mediates a malate-dependent inward-rectifying current, whereas the ma1-mediated transmembrane current is much weaker, indicating that ma1 has significantly lower malate transport activity than Ma1. RNA interference suppression of <i>Ma1</i> expression in 'McIntosh' apple leaves, 'Empire' apple fruit, and 'Orin' apple calli results in a significant decrease in malate level. Genotyping and phenotyping of 186 apple accessions from a diverse genetic background of 17 <i>Malus</i> species combined with the functional analyses described above indicate that Ma1 plays a key role in determining fruit acidity and that the truncation of Ma1 to ma1 is genetically responsible for low fruit acidity in apple. Furthermore, we identified a C-terminal domain conserved in all tonoplast-localized ALMTs essential for Ma1 function; protein truncations into this conserved domain significantly lower Ma1 transport activity. We conclude that the truncation of Ma1 to ma1 reduces its malate transport function by removing a conserved C-terminal domain, leading to low fruit acidity in apple.