Synergism and antagonism: Unraveling the regulatory network of perianth coloration in Dendrobium nobile Lindl.

Dendrobium nobile Lindl. is a multifunctional orchid species whose perianth diversity is particularly valued in horticulture, yet the molecular mechanisms underlying perianth coloration remain elusive. Here, we compared wild-type (WT), natural mutant with lip-like petals and a purplish-red blotch at the petal base (SC), and natural mutant with lip-like lateral sepals displaying a purplish-red blotch at the sepal base and a petal-like dorsal sepal (DB) of D. nobile to investigate perianth coloration patterns via pigment analysis, transcriptomics, and functional assays. Qualitative and quantitative analysis of pigments revealed that during development, perianth pigmentation shifted from chlorophyll to anthocyanin dominance. DnMYB1, DnMYB2, DnAGL6-2 and DnSEP3 promoted anthocyanin synthesis, as validated by heterologous transient overexpression and virus-induced gene silencing (VIGS). Transcriptomics, yeast one-hybrid (Y1H), and yeast two-hybrid (Y2H) assays revealed a multi-layered regulatory mechanism: DnAGL6-2 and DnSEP3 can directly activate the anthocyanin biosynthesis pathway (ABP) genes DnF3H, DnFLS, and DnBZ1; the spatially specific distribution of DnMYB1 and DnMYB2, the synergistic interaction between DnAGL6-2 and DnMYB1, and the antagonistic interaction between DnAGL6-2 and DnSEP3 collectively determine the coloration difference between the blotch and non-blotch regions. Our study uncovers a regulatory network for perianth coloration driven by the synergistic and antagonistic interactions among transcription factors (TFs), providing new gene targets and strategies for orchid color breeding.