Dendrobium officinale, a valuable medicinal plant, contains bioactive mannan polysaccharides that exert significant health-promoting effects in humans and serve as key quality markers for D. officinale products. However, the regulatory mechanisms underlying bioactive polysaccharide biosynthesis in plants remain poorly understood. In this study, we identified an anthocyanin-specific regulator, DoMYB75, as a key transcriptional activator of mannan polysaccharide biosynthesis in D. officinale. We demonstrated that DoMYB75 directly binds to the promoters of CELLULOSE SYNTHASE-LIKE A genes (DoCSLAs) and activate their expression. Genetic evidence showed that DoMYB75 silencing reduced mannose and glucose content of water-soluble polysaccharides (WSPs) and downregulated DoCSLAs expression, whereas DoMYB75 overexpression significantly increased these monosaccharide levels and upregulated DoCSLAs expression. Interestingly, Ubi:DoMYB75 transgenic transformants exhibited enhanced anthocyanin accumulation. Further investigation revealed that DoMYB75 promotes anthocyanin biosynthesis by directly binding to and activating the DoANS promoter. Additionally, DoMYB75 overexpression markedly improved total antioxidant capacity and drought tolerance. Our findings provide novel insights into the dual regulatory role of MYB transcription factors in coordinating polysaccharide and anthocyanin biosynthesis, as well as the adaptive mechanisms of Dendrobium orchids under drought stress.

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