Structural feature of RrGGP2 promoter and functional analysis of RrNAC56 regulating RrGGP2 expression and ascorbate synthesis via stress-inducible cis-elements in Rosa roxburghii Tratt

Rosa roxburghii Tratt is a well-known horticultural crop that produces fruits with extremely high l-ascorbic acid (AsA) levels, and GDP-l-galactose phosphorylase2 (RrGGP2) encodes a major enzyme operating in AsA biosynthesis. This study aims to elucidate the transcriptional mechanism of RrGGP2 underlying AsA overproduction under abiotic stress. Herein, the sequence of RrGGP2 promoter (PRrGGP2) was isolated. The analysis of the PRrGGP2 detected an upstream open reading frame encoding a 64-amino acid peptide as well as a number of cis-acting elements responsive to environmental factors and hormones. Several truncated promoter fragments were constructed for dual-luciferase assays which revealed a critical promoter region (-1949 to -2089 bp) for PRrGGP2 activity. Overexpressing β-glucuronidase (GUS) and RrGGP2 under the control of PRrGGP2 in transgenic Arabidopsis thaliana increased the GUS activity and AsA content, respectively. Furthermore, the extent of the increases was significantly influenced by temperature and abscisic acid. Yeast one-hybrid and dual-luciferase assays indicated that RrNAC56 could activate PRrGGP2. Cold stress significantly increased the transcription of RrNAC56 and RrGGP2 in R. roxburghii fruits, which resulted in AsA accumulation. These findings offer a theoretical foundation for understanding the transcriptional regulation of RrGGP2, while also uncover a novel mechanism of RrNAC56-RrGGP2 module-mediated abiotic stress response via regulating AsA synthesis.