Elucidation and functional characterization of natural sweetener biosynthesis in hortensia species

Among the various coumarins found in Hydrangea species, the bioactive dihydroisocoumarin known as phyllodulcin (PD) stands out as a non-caloric, high-intensity sweetener, which is up to 800 times sweeter than sucrose. Additionally, PD possesses notable medicinal properties, including antifungal, anti-ulcer, and anti-inflammatory effects. PD also plays several plant-specific roles, such as defending against phytopathogens, responding to abiotic and biotic stresses and regulating hormones. However, the biological function and biosynthetic pathway of PD in Hydrangea remain unexplored. This study employs targeted metabolomic and transcriptomic approaches to uncover the regulatory mechanisms and identify potential candidate genes involved in the biosynthesis of PD. 14 out of 182 different Hydrangea accessions, exhibiting varying levels of PD, were selected for detailed analysis. Accessions of H. macrophylla with high PD levels displayed distinct metabolite profiles compared to those with low PD concentrations. Specifically, pathways involving caffeic acid, ferulic acid, and their derivatives, such as scopolin, scopoletin, esculetin, and fraxetin, were predominant in accessions with low PD concentrations. Conversely, the metabolism of phenylalanine, umbelliferone, p-coumaric acid, naringenin, resveratrol, and Thn C was more active in accessions containing PD. Correlation analysis between PD and these metabolites offered valuable insights into their interrelationships. Transcriptome analysis revealed specific genes involved in phenylpropanoid biosynthesis, flavonoid biosynthesis, and stilbene biosynthesis pathways that are crucial for PD biosynthesis. Moreover, the identification of cyclase and ketoreductase genes, which were up regulated in accessions with high PD, provided further understanding of the biosynthetic pathway leading to PD. Finally, based on metabolite levels and gene expression data, a hypothetical pathway from resveratrol to Thn C was proposed, suggesting its potential involvement in PD biosynthesis.