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

Abstract

Malate accumulation in the vacuole largely determines apple (Malus domestica) fruit acidity, and low fruit acidity is strongly associated with truncation of Ma1, an ortholog of ALUMINUM-ACTIVATED MALATE TRANSPORTER9 (ALMT9) in Arabidopsis (Arabidopsis thaliana). A mutation at base 1,455 in the open reading frame of Ma1 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 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 that ma1 has significantly lower malate transport activity than Ma1. RNA interference suppression of Ma1 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 Malus 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.

BTB-BACK-TAZ domain protein MdBT2-mediated MdMYB73 ubiquitination negatively regulates malate accumulation and vacuolar acidification in apple

“…Malate is a pivotal metabolite that is involved in regulating osmotic pressure, pH homeostasis, and stress resistance, and it significantly influences fruit quality 2,7 . A previous study has shown that the BTB-BACK-TAZ protein MdBT2 ubiquitinates the bHLH TF MdCIbHLH1 to regulate malate accumulation and vacuolar acidification 24 .…”

“…However, whether MdBT2 affects malate accumulation through MdMYB1 still needs further study. The role of MdALMT9 in regulating malate accumulation has been clearly demonstrated 2,5,7,20 , and MdMYB73 regulates malate accumulation by activating the transcription of MdALMT9 (ref. 2 ).…”

“…Malate, a major intermediate in several metabolic processes, is present in most plant species and plays critical roles in regulating osmotic pressure, pH homeostasis, nutrient absorption, and stress resistance 1 , 2 . Malate also acts as an important acidity indicator for fruit quality that influences the perception of its sweetness, the accumulation of other nutrients, and the processing quality of fruit wine and juice 3 – 7 . Malate metabolism is a complex biological process and is comprised of synthesis, degradation, and transport 7 , 8 .…”

Identification of two QTLs associated with high fruit acidity in apple using pooled genome sequencing analysis

“…The success could be attributed to the choice of the mapping population GMAL 4595 and the adaptation to the AFDDD mapping approach. Population GMAL 4595 was previously used for fine genetic mapping of Ma, leading to the identification of the critical premature stop codon mutation in Ma1 that was causal for low acidity 4,14,17 . One of the important characteristics of population GMAL 4595 was the non-detectable effect of Ma3 on chromosome 8, making it highly suitable for detection of other genes controlling high acidity in the background of Ma-.…”

“…The genetic cause for low acidity had been attributed to the premature stop codon leading mutation from G to A at base 1455 in the Ma1 open reading frame (ORF), which effectively truncates 84 amino acids at the MA1 C-terminus, presumably creating a malfunctioned MA1 14 . Indeed, a latest study demonstrates that the C-terminus of MA1 is essential for malate transport as the Ma1-1455A (allele ma1) protein showed significantly lower malate transport activity than the Ma1-1455G (Ma1) protein when expressed in both Xenopus laevis oocytes and Nicotiana benthamiana cells 17 .…”