Objective To investigate the regulatory mechanisms by which different cultivation substrates influence the accumulation of pharmacologically active components in near-wild Tiepishihu (Dendrobium officinale), and provide a basis for elucidating the molecular mechanisms underlying quality differences and for optimizing cultivation substrates. Methods D. officinale samples grown on stone-based and wood-based substrates were used as experimental materials. Non-targeted metabolomics was employed to identify and compare the secondary metabolite profiles under the two cultivation modes, while transcriptome sequencing was conducted to screen differentially expressed genes (DEGs). KEGG and other databases were used for pathway enrichment analysis, which was then integrated with metabolite variation data. Results A total of 2 878 metabolites were identified by metabolomics, of which 354 showed significant differences between the two cultivation modes. These metabolites were mainly involved in phenylpropanoids, flavonoids, alkaloids, lipids, and aromatic amino acids. Flavonoids, alkaloids, and certain amino acid-derived metabolites exhibited overall higher accumulation in stone-grown samples, whereas lipids and steroid-related compounds were relatively enriched in wood-grown samples. Transcriptome sequencing identified 2 966 DEGs. KEGG enrichment analysis indicated significant differentiation in pathways including phenylpropanoid biosynthesis (ko00940), flavonoid biosynthesis (ko00941), phenylalanine/tyrosine/tryptophan biosynthesis (ko00400), and tropane, piperidine and pyridine alkaloid biosynthesis (ko00960). Genes encoding key enzymes in flavonoid biosynthesis [such as phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), flavanone 3-hydroxylase (F3H)] were upregulated in stone-grown samples, consistent with the accumulation patterns of the corresponding metabolites. Integrated analysis suggested that cultivation substrates may regulate the expression of genes involved in phenylpropanoid-flavonoid-alkaloid metabolism, thereby altering the direction of metabolic flux allocation and ultimately shaping distinct metabolic phenotypes. Conclusion Cultivation substrates influence the types and accumulation levels of active constituents in D. officinale by regulating the expression of genes in phenylpropanoid, flavonoid, and alkaloid biosynthetic pathways, resulting in quality differences between stone-grown and wood-grown materials. This study systematically elucidates the molecular mechanisms underlying quality variation in D. officinale and provides a theoretical basis for cultivation substrate optimization and graded utilization of medicinal materials.

Dendrobium officinale Kimura & Migo  /  non-targeted metabolomics  /  transcriptomics  /  cultivation substrate  /  pharmacologically active components