Deciphering differentially expressed genes and a flavonoid analysis reveal that the metabolic flux shift of flavonoids lightens flower coloration in γ-irradiation-induced Dendrobium hybrid mutants

Flower coloration, a key ornamental trait in Dendrobium orchids, is primarily determined by anthocyanin and flavonoid metabolism. Although the core anthocyanin biosynthetic pathway and its transcriptional regulation have been extensively studied in model plants and natural orchid variants, the molecular basis of pigmentation changes induced by physical mutagens such as γ-irradiation remains poorly understood. In this study, we analyzed three γ-irradiation-induced Dendrobium hybrid mutants exhibiting distinct floral pigmentation patterns, namely, white-centered pink (S29), fully white (S30), and white-edged pink (S34). Integrated RNA sequencing and metabolite profiling revealed that these pigmentation differences were closely associated with anthocyanin abundance and broad transcriptional reprogramming of the flavonoid biosynthetic pathway. In the mutants S29 and S30, early biosynthetic genes such as chalcone synthase (CHS) and flavanone 3-hydroxylase (F3H) were markedly downregulated, whereas flavonoid 3’-hydroxylase (F3’H) and anthocyanidin reductase (ANR) were strongly upregulated. Consistently, metabolite analyses showed the severe depletion of cyanidin-based anthocyanins, accompanied by abnormal accumulation of quercetin derivatives and p-coumaroyl-glucose conjugates. Notably, the stress-responsive transcription factor WRKY70 exhibited exceptionally high expression in S29 and S30, coinciding with ANR activation and anthocyanin reduction. In contrast, S34 retained partial pigmentation and accumulated a distinct quercetin-O-rutinoside isomer, indicating a pastel-like coloration and milder redistribution of flavonoid metabolic flux. Together, these results suggest that γ-irradiation can reshape flower coloration in Dendrobium through the redistribution of flavonoid metabolic flux rather than simple disruption of canonical anthocyanin biosynthetic genes. Although further genome-level and targeted metabolite analyses will be required to elucidate the precise genetic determinants, this study highlights the plasticity of the flavonoid metabolism under irradiation-induced perturbations and demonstrates the utility of irradiation mutagenesis as a non-transgenic strategy for the generation of novel ornamental flower traits in orchids.