CcMYB24 redirects flavonoid metabolic flux toward flavonols by activating CcFLS and CcCHI to drive leaf variegation in Cymbidium

Leaf color polymorphism strongly influences the ornamental and commercial value of orchids, yet the transcriptional mechanisms that partition flavonoid flux between flavonols and anthocyanins in variegated leaves remain poorly resolved, especially in monocots. Based on our previous findings in γ-ray-induced hybrid Cymbidium cultivars, the R2R3-MYB transcription factor CcMYB24 was identified as a candidate regulator. Its transcript levels were selectively enhanced in the yellow leaf sectors and showed positive correlation with flavonol content, suggesting its potential role in the metabolic partitioning of flavonoids. To functionally test the regulatory capacity of CcMYB24 in a tractable orchid system, we established an optimized Agrobacterium-mediated protocorm transformation protocol in Dendrobium officinale (transformation efficiency ~ 2.1%) and generated CcMYB24-overexpression lines. In D. officinale, ectopic expression of CcMYB24 resulted in substantial increases in flavonol (~ 2-fold) and total flavonoid contents without significantly altering chlorophyll or carotenoid levels, producing a stable yellow-green foliar phenotype (RHS Green Group 143). Dual-luciferase reporter assays showed that CcMYB24 activates the promoters of CcFLS and CcCHI, while yeast one-hybrid and EMSA assays revealed preferential binding to the MYB core motif 5′-CTACC-3′. UV-B (Ultraviolet-b radiation) irradiation further enhanced this regulatory module by elevating CcMYB24 transcript abundance and amplifying flavonol accumulation. Our study establishes the first monocot model demonstrating that a single MYB transcription factor (CcMYB24) orchestrates spatial leaf variegation through coordinated transcriptional activation and preferential flux partitioning, defining a CcMYB24-CcFLS/CcCHI regulatory module with conserved capacity to bias flavonoid output toward flavonols in orchids. These findings provide new insights into the regulatory basis of flavonoid flux partitioning in planta and offer a molecular tool for precision breeding of patterned orchid cultivars.