Combined Metabolomic and Transcriptomic Analyses Reveal the Fruit Color Mutation in Ilex rotunda

Ilex rotunda Thunb. is a prestigious ornamental tree renowned for its vibrant red fruits, yet the molecular mechanisms governing its fruit color variation remain poorly understood. The discovery of a rare yellow-fruited natural bud sport cultivar, ‘Peace Time’, provides an ideal model to investigate these processes compared to the wild-type red fruit. In this study, we integrated physiological evaluations, untargeted metabolomics, and de novo transcriptomics across multiple fruit developmental stages to elucidate the basis of this color transition. Our results demonstrated that the yellow phenotype is characterized by high lightness and yellowness values, driven by the profound suppression of anthocyanin biosynthesis. Biochemical and transcriptomic profiling revealed that DFR (dihydroflavonol 4-reductase), a critical “gatekeeper” gene, experiences severe transcriptional silencing in the yellow-fruited cultivar. This enzymatic bottleneck triggers a “passive substrate overflow,” redirecting shared precursors toward the parallel flavonol branch, resulting in the substantial accumulation of specific flavonols, including rutin and isoquercitrin. Furthermore, correlation network analysis highlighted a putative dual regulatory module associated with this metabolic reprogramming: the down-regulation of the putative activator bHLH30 coupled with the robust up-regulation of the putative repressor bHLH51, together likely contributing to the silencing of DFR transcription. These findings provide a comprehensive “dual-module” and “passive overflow” framework for fruit coloration in I. rotunda, highlighting a remarkable metabolic plasticity that reshapes this cultivar’s phytochemical profile and offers vital insights for future ornamental breeding.