Apple ALMT9 requires a conserved C-terminal domain for malate transport underlying fruit acidity

Authors: LI, CHUNLONG - Cornell University; DOUGHERTY, LAURA - Cornell University; COLUCCIO, ALISON - Former ARS Employee; MENG, DONG - Cornell University; EL-SHARKWY, ISLAM - Cornell University; BOREJSZA-WYSOCKA, EWA - Cornell University; LIANG, DONG - Cornell University; Pineros, Miguel; XU, KENONG - Cornell University; CHENG, LAILIANG - Cornell University

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/18/2019
Publication Date: 2/12/2020
Citation: Li, C., Dougherty, L., Coluccio, A., Meng, D., El-Sharkwy, I., Borejsza-Wysocka, E., Liang, D., Pineros, M., Xu, K., Cheng, L. 2020. Apple ALMT9 requires a conserved C-terminal domain for malate transport underlying fruit acidity. Plant Physiology. 182(2):992-1006. https://doi.org/10.1104/pp.19.01300.
DOI: https://doi.org/10.1104/pp.19.01300

Interpretive Summary: Although improvement of apple fruit quality has been one of the major goals in apple breeding programs, fruit quality is complex and comprises many traits, including fruit size, texture, fruit acidity, soluble contents and, others. The incomplete understanding of the genetics and cellular mechanisms determining apple fruit quality continues to make genetic improvement of fruit challenging for apple breeders. In this work, we demonstrate that Ma1, and apple gene previously associated with apple fruit acidity, encodes a protein that mediates the transport and accumulation of the malate in the apple fruit. The accumulation of this organic acid in the vacuoles of the fruit cells largely determines the degree of apple fruit acidity. We have also shown that the low fruit acidity associated with the recessive ma1 allele, is due to the functional disruption of the Ma1 protein, thereby impeding malate accumulation. The mechanistic findings in this work should assist selection in apple breeding programs targeting the fruit acidity trait.

Technical Abstract: Malate is not only a key metabolite involved in glycolysis and Krebs cycle, but its accumulation in the vacuole also largely determines apple fruit acidity. Our earlier work showed that a mutation at base 1455 in the open reading frame of Ma1, an ortholog of ALUMINUM-ACTIVATED MALATE TRANSPORTER 9 (ALMT9) in Arabidopsis, leads to a premature stop codon that truncates the protein by 84 amino acids at its C-terminal. This truncation is strongly associated with low fruit acidity in apple. Here, we report that both the full length protein, Ma1, and its naturally occurring truncated protein, ma1, localize to the tonoplast; when expressed in Xenopus laevis oocytes and Nicotiana benthamiana cells, Ma1 mediates a malate-dependent inward-rectifying current whereas the ma1-mediated transmembrane current is much weaker, indicating ma1 has a significantly lower malate transport activity than Ma1. RNAi suppression of Ma1 expression in transgenic 'McIntosh' apple plants 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 Malus species combined with the functional analyses described above indicate that Ma1 plays a key role in determining fruit acidity and the truncation of Ma1 into 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 leads to a significant reduction in Ma1 transport activity.