RcDHD/SDH gene family differentially drive gallic and ellagic acid biosynthesis in Rubus chingii fruit: insights from transient overexpression and metabolic profiling

The 3-dehydroquinic acid dehydrogenase/shikimate dehydrogenase ( DHD/SDH ) gene, which encodes a protein, acts as a dual-enzyme capable of catalyzing both shikimate and gallic acid synthesis. This dual-enzyme is playing a pivotal regulatory role in the gallic acid metabolic pathway. Objective: To investigate the functional roles of DHD/SDH in the gallic acid and ellagic acid synthesis pathways within the fruit of Chinese raspberry ( Rubus chingii Hu). Method: Using Rubus chingii Hu ( R. chingii ) as experimental material, Agrobacterium-mediated transformation was utilized to introduce over-expression vectors carrying the target gene. Specifically, Agrobacterium cultures harboring the R. chingii RcDHD/SDH target gene over-expression vectors ranging from pCAMBIA1300-RcDHD/SDH1 to RcDHD/SDH4-GFP were injected into raspberry fruit tissue for transient expression. To evaluate the impact, fluorescent quantitative PCR was employed to assess DHD/SDH gene expression levels, while HPLC was used to determine gallic acid and ellagic acid content, providing preliminary insights into the biological roles of the RcDHD/SDH gene family. Results: Subcellular localization experiments revealed that these gene products are localized to the nucleus. This gene cluster is pivotal in controlling the synthesis of phenolic acid in R. chingii through temporal expression patterns, with its activity aligning closely with the level of ellagic acid and gallic acid. In the case of thornless cultivars, RcDHD/SDH1 - RcDHD/SDH2 take center stage, enabling an early rapid accumulation, whereas thorny cultivars activate RcDHD/SDH3 - RcDHD/SDH4 to implement a sustained synthesis approach during late-stage, highlighting gene functional differentiation in secondary metabolic strategies between cultivars. Conclusion: RcDHD/SDH stimulates biosynthesis and accumulation of gallic and ellagic acid in R. chingii , providing a theoretical basis for molecular breeding to enhance fruit ellagic acid content.