Chunmei Zhong

Although steroidal saponins from Dioscorea species have substantial economic value and versatile utilization potential, the evolutionary origin and regulatory mechanisms underlying their biosynthesis remain poorly understood. Here, we conducted a comparative genomic analysis across representative plant lineages ranging from algae to angiosperms to examine the copy number variation and syntenic organization of steroidal saponin biosynthetic genes. Our results reveal that ancestral sterol biosynthetic components were already present in bryophytes. However, synteny analysis indicates that stable genomic integration of these genes was established no earlier than in pteridophytes. By integrating transcriptome–metabolome association analyses with branch-site selection tests, we identified strong positive selection acting on DcWRKY33 within the Dioscorea lineage, suggesting adaptive evolution and highlighting the importance of regulatory innovation in steroidal saponin diversification. Functional analyses demonstrated that DcWRKY33 functions as a positive regulator of steroidal saponin biosynthesis: transient overexpression upregulates the expression of structural genes and increases saponin accumulation, whereas gene silencing produced opposite effects. Mechanistically, dual-luciferase and yeast one-hybrid assays indicated that DcWRKY33 activates DcHDR and DcCYP51, while indirectly regulating DcHMGR through modulation of DcWRKY11, thereby orchestrating biosynthesis across both the MEP and MVA pathways. Promoter analysis revealed W-box motifs partially absence in non-saponin-producing Dioscorea, and heterologous expression in tobacco confirmed lineage-specific regulatory capacity despite conserved cis-elements. Collectively, our study links pathway evolution with regulatory innovation and establishes a mechanistic framework for understanding the evolution and transcriptional control of steroidal saponin biosynthesis in D. composita, providing insights for metabolic improvement of medicinal and horticultural yam species.

DcMYB5 regulates diosgenin biosynthesis in Dioscorea composita by binding to the DcCYP90B promoter and repressing its transcription. Diosgenin is an important bioactive metabolite in Dioscorea with significant pharmaceutical and ecological value. However, the transcriptional regulatory mechanisms of its biosynthesis remain poorly understood. Here, we identified and characterized an SG5 subgroup R2R3-MYB transcription factor (TF) from Dioscorea composita, designated DcMYB5. Sequence and phylogenetic analyses identified DcMYB5 as a MYB5-type SG5 MYB TF with typical R2R3 conserved domains but lacking the C3 motif at the C-terminus. Functional gene silencing and overexpression experiments validated that DcMYB5 modulates the biosynthesis of diosgenin and its derivatives, along with tuber growth. Dual-luciferase and yeast one-hybrid assays further indicated that DcMYB5 binds to the DcCYP90B promoter and represses its transcription, thereby regulating diosgenin accumulation. Overall, this study provides new insights into how R2R3-MYB TFs coordinate steroidal metabolism with growth in D. composita and delivers an actionable molecular target for agricultural biotechnology to enhance high-value steroidal saponin production.

Transforming lignocellulose into high-value biochemicals via microbial fermentation is critical for valorizing agricultural/forestry residues and advancing sustainable biomanufacturing. However, fermentation efficiency is hindered by environmental stressors. Herein, soluble lignin was prepared by alkaline dissolution and dialysis of enzymatic hydrolysis lignin and its effects on the above three fermentations were systematically investigated. Results showed that soluble lignin buffered fermentation pH via carboxyl protonation, scavenged ROS through phenolic hydroxyl groups to alleviate microbial inactivation, and chelated heavy metals (e.g., Pb²⁺, Cd²⁺) via phenolic hydroxyl/carboxyl coordination to reduce toxicity. Notably, it exhibited pH-responsive recyclability: dissolving at neutral pH (initial fermentation) and precipitating at acidic pH (fermentation end), enabling centrifugal recovery and reuse for ≥10 cycles with retained performance. This study demonstrates that soluble lignin universally enhances multiple microbial fermentations via physicochemical synergies, providing a theoretical basis for optimizing lignocellulose-based fermentation and new avenues for high-value lignin utilization in biomanufacturing.

Proanthocyanidins (PAs) are condensed tannins with a wide range of pharmacological properties and have a variety of nutritional roles in food processing and medicinal supplements. Although studies have shown that methyl jasmonate (MeJA) promotes the accumulation of PAs, the underlying molecular regulatory network remains unclear. Our study herein indicated that MeJA also dramatically induces the accumulation of PAs in Dioscorea composita. Comparative transcriptomic and metabolomic analyses were further employed to investigate its detailed molecular regulatory network. In addition, we further identified a classical WRKY transcription factor named DcWRKY11 and characterized its function in regulating MeJA‐induced PAs biosynthesis. Silencing of DcWRKY11 in D. composita decreased PAs accumulation, whereas heterologous overexpression of DcWRKY11 in Arabidopsis thaliana increased PAs accumulation. Further evidence verified that DcWRKY11 directly binds to the promoter of AtTT2 thereby regulates the expression of the PAs biosynthetic genes in A. thaliana. These results suggest that DcWRKY11 functions as a positive regulator in the regulation of PAs biosynthesis. Our findings not only provide a deeper understanding of the regulatory network of MeJA‐mediated PAs biosynthesis in D. composita, but also provide abundant candidate genes for further investigation of the regulatory mechanism of PAs biosynthesis.

Key message DcWRKY5 increases the antioxidant enzyme activity and proline accumulation, oppositely, reduces the accumulation of ROS and MDA, through directly activating the genes expression, finally enhances the salt and drought tolerance. Abstract Drought and salinity are two main environmental factors that limit the large-scale cultivation of the medicinal plant Dioscorea composita (D. composita). WRKY transcription factors (TFs) play vital roles in regulating drought and salt tolerance in plants. Nevertheless, the molecular mechanism of WRKY TF mediates drought and salt resistance of D. composita remains largely unknown. Here, we isolated and characterized a WRKY TF from D. composita, namely DcWRKY5, which was localized to the nucleus and bound to the W-box cis-acting elements. Expression pattern analysis showed that it was highly expressed in root and significantly up-regulated in the presence of salt, polyethylene glycol-6000 (PEG-6000) and abscisic acid (ABA). Heterologous expression of DcWRKY5 increased salt and drought tolerance in Arabidopsis, but was insensitive to ABA. In addition, compared with the wild type, the DcWRKY5 overexpressing transgenic lines had more proline, higher antioxidant enzyme (POD, SOD, and CAT) activities, less reactive oxygen species (ROS) and malondialdehyde (MDA). Correspondingly, the overexpression of DcWRKY5 modulated the expression of genes related to salt and drought stresses, such as AtSS1, AtP5CS1, AtCAT, AtSOD1, AtRD22, and AtABF2. Dual luciferase assay and Y1H were further confirmed that DcWRKY5 activate the promoter of AtSOD1 and AtABF2 through directly binding to the enrichment region of the W-box cis-acting elements. These results suggest that DcWRKY5 is a positive regulator of the drought and salt tolerance in D. composita and has potential applications in transgenic breeding.