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Background Astragalus membranaceus var. mongholicus (Astragalus), acknowledged as a pivotal “One Root of Medicine and Food”, boasts dual applications in both culinary and medicinal domains. The growth and metabolite accumulation of medicinal roots during the harvest period is intricately regulated by a transcriptional regulatory network. One key challenge is to accurately pinpoint the harvest date during the transition from conventional yield content of medicinal materials to high and to identify the core regulators governing such a critical transition. To solve this problem, we performed a correlation analysis of phenotypic, transcriptome, and metabolome dynamics during the harvesting of Astragalus roots. Results First, our analysis identified stage-specific expression patterns for a significant proportion of the Astragalus root genes and unraveled the chronology of events that happen at the early and later stages of root harvest. Then, the results showed that different root developmental stages can be depicted by co-expressed genes of Astragalus. Moreover, we identified the key components and transcriptional regulation processes that determine root development during harvest. Furthermore, through correlating phenotypes, transcriptomes, and metabolomes at different harvesting periods, period D (Nov.6) was identified as the critical period of yield and flavonoid content increase, which is consistent with morphological and metabolic changes. In particular, we identified a flavonoid biosynthesis metabolite, isoliquiritigenin, as a core regulator of the synthesis of associated secondary metabolites in Astragalus. Further analyses and experiments showed that HMGCR , 4CL , CHS , and SQLE , along with its associated differentially expressed genes, induced conversion of metabolism processes, including the biosynthesis of isoflavones and triterpenoid saponins substances, thus leading to the transition to higher medicinal materials yield and active ingredient content. Conclusions The findings of this work will clarify the differences in the biosynthetic mechanism of astragaloside IV and calycosin 7-O-β-D-glucopyranoside accumulation between the four harvesting periods, which will guide the harvesting and production of Astragalus.

Summary Although plant secondary metabolites are important source of new drugs, obtaining these compounds is challenging due to their high structural diversity and low abundance. The roots of Astragalus membranaceus are a popular herbal medicine worldwide. It contains a series of cycloartane‐type saponins (astragalosides) as hepatoprotective and antivirus components. However, astragalosides exhibit complex sugar substitution patterns which hindered their purification and bioactivity investigation. In this work, glycosyltransferases (GT) from A. membranaceus were studied to synthesize structurally diverse astragalosides. Three new GTs, AmGT1/5 and AmGT9, were characterized as 3‐O‐glycosyltransferase and 25‐O‐glycosyltransferase of cycloastragenol respectively. AmGT1G146V/I variants were obtained as specific 3‐O‐xylosyltransferases by sequence alignment, molecular modelling and site‐directed mutagenesis. A combinatorial synthesis system was established using AmGT1/5/9, AmGT1G146V/S and the reported AmGT8 and AmGT8A394F. The system allowed the synthesis of 13 astragalosides in Astragalus root with conversion rates from 22.6% to 98.7%, covering most of the sugar‐substitution patterns for astragalosides. In addition, AmGT1 exhibited remarkable sugar donor promiscuity to use 10 different donors, and was used to synthesize three novel astragalosides and ginsenosides. Glycosylation remarkably improved the hepatoprotective and SARS‐CoV‐2 inhibition activities for triterpenoids. This is one of the first attempts to produce a series of herbal constituents via combinatorial synthesis. The results provided new biocatalytic tools for saponin biosynthesis. Glycosylation of various bioactive astragalosides.

Powdery mildew is one of the major diseases affecting Astragalus membranaceus var. mongholicus (Bunge) P. K. Hsiao (Am) , yet the molecular mechanisms underlying its defense response to this pathogen remain unclear. To identify candidate genes and differential biomarkers involved in resistance to powdery mildew, we used a highly resistant Am germplasm (202302006) selected from previous studies. After natural disease inoculation in the field, transcriptomic and metabolomic sequencing were performed. Differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) in roots at various time points in response to powdery mildew were identified. Through DEG analysis, WGCNA, and LASSO regression, candidate genes and differentially abundant metabolites related to powdery mildew resistance were obtained. 6 upregulated candidate genes were enriched in pathways such as lipoic acid metabolism, sphingolipid metabolism, and carbon metabolism. 8 differential biomarkers were selected, with L-tartaric acid and ornithine identified as potential regulatory targets. Integrated omics analysis revealed significant enrichment of DEGs and DAMs in specific metabolic and biosynthetic pathways, with some metabolites showing positive/negative correlations with candidate genes, highlighting two key regulatory pathways. This study provides a comprehensive analysis of the mechanisms underlying the resistance of Am to powdery mildew, offering theoretical and technical support for the breeding of new powdery mildew-resistant cultivars.

Background WRKY transcription factors (TFs) are important transcriptional regulators in plants, with their members widely involved in plant growth and development as well as responses to abiotic stresses. However, researches on WRKY genes in the medicinal plant A. membranaceus are scarce. Specifically, the roles of AmWRKYs in cold stress adaptation and their regulatory effects on flavonoid biosynthesis, which determines both medicinal quality and stress resistance, remain largely unexplored. Given its high economic value and extreme sensitivity to cold in its main cultivation regions, identifying key regulators of its cold tolerance is crucial for genetic improvement. Result In this study, 94 AmWRKY were identified based on genome analysis, distributed across 8 chromosomes. AmWRKYs are structurally conserved, all carrying the core conserved domain \"WRKYGQK\" and classified into 6 subgroups. Cis-acting elements responsive to plant growth and development, abiotic stress, and hormone responses were identified in the promoter regions. Additionally, the transcriptome and metabolome data under cold stress were analyzed, and a co-expression and metabolite association network of AmWRKY genes was constructed. Sixteen AmWRKY transcription factors showed dynamic expression under cold stress, among which AmWRKY22/24/44/65 were continuously upregulated, indicating their core roles in cold adaptation. Co-expression network analysis revealed the synergistic effects of AmWRKY with AP2/ERF, MYB, and NAC transcription factors, forming a regulatory module integrating hormone signaling, antioxidant pathways, and circadian rhythm regulation. Metabolomics analysis indicated that AmWRKY24/44 expression was positively correlated with the upregulation of key flavonoid biosynthesis genes ( AmCHS , AmFLS ) and the accumulation of nine cold-responsive flavonoids. These findings suggest a new regulatory pathway of AmWRKY24/44  → flavonoid biosynthesis → cold resistance, linking secondary metabolism with environmental adaptation. Conclusion This study reveals a novel regulatory pathway—“ AmWRKY24/44 ” → flavonoid biosynthesis → cold resistance—in A. membranaceus , providing deeper mechanistic insights into how WRKY transcription factors modulate secondary metabolism under cold stress. These findings offer a valuable theoretical foundation for genetic improvement of cold tolerance in this medicinally important species.

Radix Astragali is commonly used in traditional Chinese medicine, and its quality is closely related to ecological factors, such as climate and soil, in the production area. To provide high-quality Radix Astragali to Chinese and foreign markets, we used maximum entropy model and statistical analysis method, combined with data on ecological factors, Astragalus membranaceus var. mongholicus geographical distribution, and index component content to predict the ecological suitability distribution of A. membranaceus var. mongholicus and establish the relationship between astragaloside IV and calycosin-7-glucoside in this species and ecological factors. Subsequently, we could determine the suitability regionalization of high-quality A. membranaceus var. mongholicus in Inner Mongolia, China. The results showed that the standard deviation of seasonal changes in temperature (40.6%), precipitation in October (15.7%), vegetation type (14.3%), soil type (9.2%), and mean sunshine duration in the growing season (9.1%) were the top five most influential factors out of the 17 main ecological factors affecting the distribution of A. membranaceus var. mongholicus . The standard deviation of seasonal changes in temperature, precipitation in October, precipitation in April, soil pH, and mean sunshine duration in the growing season were found to be the key ecological factors affecting the accumulation of astragaloside IV and calycosin-7-glucoside in A. membranaceus var. mongholicus . The regions with the highest-quality A. membranaceus var. mongholicus were distributed in Baotou (Guyang County), Hohhot (Wuchuan County), and central Wulanchabu (Chahar Right Middle Banner, Chahar Right Back Banner, and Shangdu County) and its surroundings in Inner Mongolia. Baotou, Hohhot, and their surrounding areas were the main traditional production areas of A. membranaceus var. mongholicus , and central Wulanchabu was a potentially suitable distribution area of this species . The main production areas were consistent with the actual production base of A. membranaceus var. mongholicus . This study therefore provides a scientific basis to guide the cultivation of A. membranaceus var. mongholicus .

Astragalus membranaceus is a well-known medicinal plant rich in bioactive compounds, particularly the isoflavonoid calycosin, which exhibits diverse pharmacological properties. However, there is limited research on the influence of light conditions on calycosin biosynthesis and the underlying molecular mechanisms. In this study, we exposed in vitro plantlets of A. membranaceus to different LED light sources (white, red, and blue) and found that red light treatment significantly enhanced both biomass accumulation and calycosin content. Through transcriptome analysis combined with real-time PCR we identified several key genes involved in calycosin biosynthesis, among which AmI3’H showed the most significant upregulation under red light. Structural analysis and molecular docking indicated that AmI3’H , a member of the CYP81 clan of cytochrome P450 enzymes, interacts with formononetin, a precursor of calycosin. To verify its functional role, transient overexpression of AmI3’H was performed in Nicotiana benthamiana using Agrobacterium -mediated infiltration along with exogenous formononetin. The co-infiltration resulted in calycosin production exclusively at 24 h post-infiltration, accompanied by elevated antioxidant activity and total flavonoid content. Additionally, transcriptome-based prediction identified AmbHLH30 as a potential transcription factor (TF) regulating calycosin biosynthesis, showing the highest expression under red light conditions. These results suggest that red light not only promotes calycosin accumulation but also modulates the expression of biosynthetic genes and transcriptional regulators, providing valuable insights for the metabolic engineering of isoflavonoid biosynthesis in A. membranaceus .

Drought and other abiotic stressors exert significant impacts on plant growth, development, and yield. WRKY transcription factors (TFs) are pivotal in regulating plant responses to such stresses. This study conducted a genome-wide identification of the Astragalus membranaceus ( A. membranaceus ) WRKY TF family, aiming at providing essential stress-tolerance genes for molecular breeding in A. membranaceus . Using bioinformatics approaches, including phylogenetic analysis, gene structure analysis, protein conserved motif prediction, and promoter cis-acting element examination, we identified 76 WRKY TF family members unevenly distributed across eight chromosomes. Phylogenetic analysis categorized the 76 AmWRKY proteins into three major classes: 14 in Class I, 52 in Class II, and 10 in Class III. Analysis of conserved structural domains revealed that similarities among domains of the same class AmWRKYs, with a predominance of members containing the conserved “WRKYGQK” heptapeptide domain and zinc finger structure, despite a few members had conserved domain variants. Gene structure analysis demonstrated that AmWRKY genes include 1–6 exons and 1–5 introns. GO and KEGG analyses revealed the differentially expressed WRKY genes are primarily transcription regulators involved in stress response pathways, with significant enrichment in DNA-binding activities, nuclear localization, and plant-pathogen/MAPK signaling. Transcriptome and qRT-PCR analyses indicated that several WRKY TFs, including AmWRKY8 , were involved in the response to drought stress. Furthermore, it was confirmed that AmWRKY8 is localized exclusively in the nucleus and possesses transcriptional activation activity. Protein interaction analysis revealed that AmWRKY8 coordinates drought stress responses by interacting with key regulators of hormone signaling (AtWRKY70, ERF6), mitochondrial stress (NAC017), and developmental processes (HBI1). These findings are useful in further investigations into the regulatory role of this this TF in abiotic stress responses.

The effect of endophytic fungal biotransformation on the formation of metabolic substances has become a non-negligible factor in assessing the quality of medicinal plants. However, the relevant evidences are still particularly lacking. In this study, an endophytic fungus CA3-A with biotransformation activity was screened and identified as Talaromyces coprophilus from root of Astragalus mongholicus . Its biotransformation effect on host metabolism was studied by co-culture with fungus and host root. The results showed that in addition to the significant changes in primary metabolites, secondary metabolite conversions are mainly focused on compounds such as terpenoids, phenols and flavonoids. The transformed metabolic pathways are mainly enriched in glycerophospholipid metabolism, ubiquinone and terpenoid quinones biosynthesis, and tyrosine metabolism. A total of 38 signature differential metabolites are found through multivariate statistical analysis. In addition to eleven primary metabolites including glycerides and glycerophospholipids, twenty-eight secondary metabolites distribute in terpenes, sterols, phenols, flavonoids, benzene, lipids and other classes of compounds, respectively. In conclusion, this study aims to quickly focus on the signature differentially metabolites from a large amount of information, narrow the possible range of potential transformation products, and provide help for understanding of influence of endophytes on host metabolism and the search for new natural products.

Astragalus membranaceus has been used in traditional Chinese medicine for over 2,000 years. Its major active triterpenoid saponins, astragalosides, have attracted great attention due to their multiple health benefits and applications in medicine. Despite this, the biosynthetic machinery for astragalosides remains enigmatic. Here a chromosome-level genome assembly of A . membranaceus was generated. The identification of two tailoring enzymes required for astragaloside biosynthesis enabled the discovery of a triterpenoid biosynthetic gene cluster, leading to elucidation of the complete astragaloside biosynthetic pathway. This pathway is characterized by a sequence of selective hydroxylation, epoxidation and glycosylation reactions, which are mediated by three cytochrome P450s, one 2-oxoglutarate-dependent dioxygenase and two glycosyltransferases. Reconstitution of this biosynthetic machinery in Nicotiana benthamiana allowed for heterologous production of astragaloside IV. These findings build a solid foundation for addressing the sourcing issues associated with astragalosides and broaden our understanding of the diversity of terpene biosynthetic gene clusters. Astragaloside IV, the key active compound in Radix Astragali, a medicinal plant used for immunomodulation in Asia and Europe, now has a fully elucidated biosynthetic pathway, enabling its efficient production through synthetic biology.

Background Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao ( A. mongolicus ) is an important traditional Chinese herb that is cultivated on a large scale in northwestern China. Understanding plant responses to drought has important effects on ecological environment recovery and local economic development. Here, we combined transcriptomics (Illumina Hiseq 2000) and metabolomics ( 1 H-NMR) to investigate how the roots of two-year-old A. mongolicus responded to 14 days of progressive drought stress. Results The dried soil reduced the relative water content (RWC) of the leaves and biomass, induced the differential expression of a large fraction of the transcriptome and significantly altered the metabolic processes. PCA analysis demonstrated that the sucrose, proline, and malate metabolites contributed greatly to the separation. Strikingly, proline was increased by almost 60-fold under severe stress compared to the control. Some backbone pathways, including glycolysis, tricarboxylic acid (TCA) cycle, glutamate-mediated proline biosynthesis, aspartate family metabolism and starch and sucrose metabolism, were significantly affected by drought. An integrated analysis of the interaction between key genes and the altered metabolites involved in these pathways was performed. Conclusions Our findings demonstrated that the expression of drought-responsive genes showed a strong stress-dose dependency. Analysis of backbone pathways of the transcriptome and metabolome revealed specific genotypic responses to different levels of drought. The variation in molecular strategies to the drought may play an important role in how A. mongolicus and other legume crops adapt to drought stress.