RroxbHLH91 positively regulates multiple genes involved in the accumulation of L-ascorbic acid

Rosa roxburghii fruit is rich in nutrients, particularly L-ascorbic acid (AsA). Although the molecular mechanisms involved in AsA accumulation in R. roxburghii have been increasingly explored, its transcriptional regulatory pathways remain poorly understood. In this study, the transcription factor RroxbHLH91 was identified as a key positive regulator of AsA accumulation through RNA-Seq and correlation analyses. The expression pattern and function of RroxbHLH91 were validated, and its target genes and regulatory network were elucidated. The results showed that RroxbHLH91 was localized in the nucleus and specifically expressed in the pulp, which contains typical conserved motif structures of the basic helix-loop-helix (bHLH) family. Transient injection and genetic transformation experiments confirmed that RroxbHLH91 positively regulates the accumulation of AsA. In addition, overexpression of RroxbHLH91 in Arabidopsis thaliana resulted in a 102% increase in leaf AsA concentration and significantly upregulated the expression of multiple genes involved in the AsA biosynthesis pathway. Furthermore, yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase reporter assays confirmed that RroxbHLH91 directly binds to the promoters of AsA biosynthesis-related genes, including RroxPME , RroxGalUR , and RroxDHAR2 , thereby positively regulating their expression. Additionally, RroxbHLH91 was found to positively regulate RroxTTG2 , which indirectly enhances the expression of these biosynthetic genes. Our findings elucidate the diverse regulatory mechanisms involved in AsA accumulation in R. roxburghii fruit and provide a theoretical foundation for future investigations into the molecular regulatory networks driving high AsA accumulation in plants.