Transcription factor MdNAC18.1 regulates malic acid accumulation in apple fruits

Malic acid, the most important organic acid component in the ripe apple fruit, is of great importance for the development of the fruit flavor and regulation of the metabolism. Previous studies have demonstrated that the P_3A-ATPase MdMa11 plays a role in determining fruit acidity, and a total of 85 positive clones were identified using yeast one-hybrid screening based on the fragment in MdMa11 promoter. Among these positive clones, the NAM domain protein was designated as MdNAC18.1. The analysis of transgenic apple calli, fruits and tomatoes indicated that MdNAC18.1 induced the organic acids accumulation to regulate fruit acidity. Luciferase (LUC) and glucuronidase (GUS) activation assays showed that MdNAC18.1 binds to the G-box motif (5'-ACGT-3') located 5227 bp upstream of transcription initiation site of the MdMa11, thereby promoting its expression. Meanwhile, the expression of MdWRKY126, MdMDH5, MdtDT, MdMYB1, and MdVHP1 was found to be significantly increased in transgenic apple calli overexpressing MdNAC18.1 and decreased in MdNAC18.1-silenced transgenic apple calli. The G-box was identified in all these five genes. However, the GUS and LUC activation assays exhibited that MdNAC18.1 activated MdWRKY126, MdMDH5, MdtDT, and MdMYB1 expression. Our findings contribute valuable insights into the complex mechanism regulating the accumulation of malate in apple fruits.