Ophiopogon japonicus, a precious medicinal plant endemic to Zhejiang Province. Its tuberous roots are rich in bioactive components such as flavonoids, possessing anti-inflammatory, antioxidant, and immunomodulatory properties. To elucidate the impact of cadmium (Cd) stress on the accumulation and biosynthetic pathway of flavonoids in O. japonicus, this study exposed O. japonicus to different concentrations of Cd stress and explored the changes through integrated transcriptomics and metabolomics analysis. The results demonstrated that Cd stress (1 mg/L and 10 mg/L) significantly increased the content of flavonoids in O. japonicus in a concentration-dependent manner. The metabolomics analysis revealed a total of 110 flavonoids including flavones, flavanols, flavonols, flavone and flavonol derivatives, flavanones, isoflavonoids, chalcones and dihydrochalcones, and anthocyanins in O. japonicus, among which flavones, flavonols, flavone and flavonol derivatives, and anthocyanins increased under Cd stress. The transcriptomics analysis identified several key flavonoid biosynthesis-associated genes with up-regulated expression under Cd stress, including 14 genes encoding 4-coumarate CoA ligase (4CL), 2 genes encoding chalcone isomerase (CHI), and 14 genes encoding phenylalanine ammonia lyase (PAL). The gene-metabolite regulatory network indicated significant positive correlations of 4CL (Cluster-21637.5012, Cluster-21637.90648, and Cluster-21637.62637) and CHI (Cluster-21637.111909 and Cluster-21637.123300) with flavonoid metabolites, suggesting that these genes promoted the synthesis of specific flavonoid metabolites, which led to the accumulation of total flavonoids under Cd stress. These findings provide theoretical support for the cultivation and utilization of medicinal plants in Cd-contaminated environments and offered new perspectives for studying plant responses to heavy metal stress.
Cadmium/toxicity* ; Flavonoids/biosynthesis* ; Metabolomics ; Ophiopogon/drug effects* ; Stress, Physiological ; Transcriptome ; Gene Expression Profiling ; Gene Expression Regulation, Plant

Soil cadmium (Cd) pollution is one of the major environmental problems globally. Ophiopogon japonicus, a multifunctional plant extensively used in traditional Chinese medicine, has demonstrated potential in environmental remediation. This study investigated the Cd accumulation pattern of O. japonicus under cadmium stress and identified the heavy metal ATPase (HMA) family members in this plant. Our results demonstrated that O. japonicus exhibited a Cd enrichment factor (EF) of 2.75, demonstrating strong potential for soil Cd pollution remediation. Nine heavy metal ATPase (HMA) members of P1B-ATPases were successfully identified from the transcriptome data of O. japonicus, with OjHMA1-OjHMA6 classified as the Zn/Co/Cd/Pb-ATPases and OjHMA7-OjHMA9 as the Cu/Ag-ATPases. The expression levels of OjHMA1, OjHMA2, OjHMA3, and OjHMA7 were significantly up-regulated under Cd stress, highlighting their crucial roles in cadmium ion absorption and transport. The topological analysis revealed that these proteins possessed characteristic transmembrane (TM) segments of the family, along with functional A, P, and N domains involved in regulating ion absorption and release. Metal ion-binding sites (M4, M5, and M6) existed on the TM segments. Based on the number of transmembrane domains and the residues at metal ion-binding sites, the plant HMA family members were categorized into three subgroups: P1B-1 ATPases, P1B-2 ATPases, and P1B-4 ATPases. Specifically, the P1B-1 ATPase subgroup included the motifs TM4(CPC), TM5(YN[X]₄P), and TM6(M[XX]SS); the P1B-2 ATPase subgroup featured the motifs TM4(CPC), TM5(K), and TM6(DKTGT); the P1B-4 ATPase subgroup contained the motifs TM4(SPC) and TM6(HE[X]GT), all of which were critical for protein functions. Molecular docking results revealed the importance of conserved sequences such as CPC/SPC, DKTGT, and HE[X]GT in metal ion coordination and stabilization. These findings provide potential molecular targets for enhancing Cd uptake and tolerance of O. japonicus by genetic engineering and lay a theoretical foundation for developing new cultivars with high Cd accumulation capacity.
Cadmium/metabolism* ; Adenosine Triphosphatases/metabolism* ; Ophiopogon/drug effects* ; Soil Pollutants/toxicity* ; Plant Proteins/metabolism* ; Stress, Physiological ; Multigene Family ; Gene Expression Regulation, Plant