Cloning, expression analysis, and subcellular position of MdPH1 related to acidity in Malus domestica Borkh

In this study, the malic acid content in Malus fruit was determined using Malus ombrophila Hand.-Mazz, with comparative transcriptome analysis conducted among developmental stages. A candidate gene for fruit acidity, designated as MdPH1, was identified. Genomic sequencing and gene structure analysis showed that the cDNA sequence contained a 2829 bp open reading frame and encoded a 942 amino acid polypeptide. The genomic DNA of MdPH1 was 4269 bp in length and consisted of eight exons and seven introns. According to the PH1 gene sequence in 10 Malus accessions, 22 single nucleotide polymorphisms (SNPs) were identified in the genomic sequence. Of these SNPs, 13 were located in introns and nine were located in exons. Variation of a SNP (G/A) on the last exon resulted in the conversion of the encoded amino acid from valine to isoleucine. In addition, the MdPH1 protein contained eight transmembrane domains (TMDs). Of these TMDs, three were located in the amino terminal and five were located in the carboxyl terminal. Exploration of the phylogenetic relationships revealed a close relationship between PH1 genes from apple and pear. Gene expression indicated that MdPH1 was highly expressed in apple fruit, followed by the leaves and flower, and finally the stem. Subcellular localization assay showed that MdPH1 was localized to the tonoplast. Our study provides useful knowledge to better understand the complex mechanisms regulating apple fruit acidity.