Citrus <scp>NAC47</scp> Modulates Vacuolar Storage of Soluble Sugars by Interacting With Type I H⁺‐Pyrophosphatase Genes Under Drought Stress

Fleshy fruit taste and quality are influenced by their soluble sugar contents. In order to improve the fruit quality, it is imperative to better understand how soluble sugars accumulate in fruit vacuoles. A previous report showed that the type I H⁺-pyrophosphatase regulates the vacuolar storage of sucrose in citrus fruits, but its transcriptional regulation still remains elusive. In this study, the NAC transcription factor gene CsNAC47 was functionally characterized and found to localize predominantly within the nucleus, consistent with its predicted role in transcriptional regulation. Moreover, drought stress markedly upregulated the transcript levels of CsNAC47 and CsVPP1/2 in Nanfeng tangerine (Citrus reticulata) fruits, indicating their potential role in sugars accumulation. Transient overexpression of CsNAC47 and CsVPP1/2 led to enhanced accumulation of soluble sugars in citrus fruit juice sacs, while virus-induced gene silencing (VIGS) of CsNAC47 and CsVPP1/2 inhibited sugar accumulation. Stable transformation in Micro-Tom tomato indicated that overexpression of CsNAC47 and CsVPP1/2 significantly elevated the vacuolar storage of soluble sugar contents. We found out through yeast one-hybrid screening assay and dual luciferase assays that CsNAC47 directly binds to the promoters of CsVPP1/2 and regulates their gene expression. This study offers novel insights into the transcriptional regulation of soluble sugar accumulation in citrus fruits under drought stress, specifically through the interaction between CsNAC47 and CsVPP1/2, which will ultimately contribute to improving fruit quality in the future.