Integrated microbiome and metabolomics analysis reveal the relationship between plant-specialized metabolites and microbial community in Phellodendron amurense.

Phellodendron amurense is the essential source of bisbenzylisoquinoline alkaloids (BIAs), making it a highly valued raw material in traditional Chinese medicine. The plant's root secondary metabolism is intricately linked to the microbial communities that surround it. However, the root-associated microbiomes of P. amurense, as well as the potential correlation between its bioactive compounds and these microbiomes, remain poorly understood. Here, the metabolic profiles of root, rhizosphere, and bulk soils of P. amurense revealed the dramatic differences in the relative content of plant-specialized metabolites. A total of 31, 21, and 0 specialized metabolites in P. amurense were identified in the root, rhizosphere soil, and bulk soil, respectively, with higher content of the seven major BIAs observed in the rhizosphere compared with that in the bulk soils. The composition of the bulk and rhizosphere microbiomes was noticeably distinct from that of the endospheric microbiome. The phylum Cyanobacteria accounted for over 60% of the root endosphere communities, and the α-diversity in root was the lowest. Targeted seven BIAs, namely, berberine, palmatine, magnocurarine, phellodendrine, jatrorrhizine, tetrahydropalmatine, and magnoflorine, were significantly positively correlated with Nectriaceae and Sphingobacteriaceae. This study has illuminated the intricate interaction networks between P. amurense root-associated microorganisms and their key chemical compounds, providing the theoretical foundation for discovering biological fertilizers and laying the groundwork for cultivating high-quality medicinal plants.

Multi-omics analysis reveals promiscuous O-glycosyltransferases involved in the diversity of flavonoid glycosides in Periploca forrestii (Apocynaceae).

Flavonoid glycosides are widespread in plants, and are of great interest owing to their diverse biological activities and effectiveness in preventing chronic diseases. Periploca forrestii, a renowned medicinal plant of the Apocynaceae family, contains diverse flavonoid glycosides and is clinically used to treat rheumatoid arthritis and traumatic injuries. However, the mechanisms underlying the biosynthesis of these flavonoid glycosides have not yet been elucidated. In this study, we used widely targeted metabolomics and full-length transcriptome sequencing to identify flavonoid diversity and biosynthetic genes in P. forrestii. A total of 120 flavonoid glycosides, including 21 C-, 96 O-, and 3 C/O-glycosides, were identified and annotated. Based on 24,123 full-length coding sequences, 99 uridine diphosphate sugar-utilizing glycosyltransferases (UGTs) were identified and classified into 14 groups. Biochemical assays revealed that four UGTs exhibited O-glycosyltransferase activity toward apigenin and luteolin. Among them, PfUGT74B4 and PfUGT92A8 were highly promiscuous and exhibited multisite O-glycosylation or consecutive glycosylation activities toward various flavonoid aglycones. These four glycosyltransferases may significantly contribute to the diversity of flavonoid glycosides in P. forrestii. Our findings provide a valuable genetic resource for further studies on P. forrestii and insights into the metabolic engineering of bioactive flavonoid glycosides.

A systematic review of ginsenoside biosynthesis, spatiotemporal distribution, and response to biotic and abiotic factors in American ginseng.

American ginseng (Panax quinquefolius) has gained recognition as a medicinal and functional food homologous product with several pharmaceutical, nutritional, and industrial applications. However, the key regulators involved in ginsenoside biosynthesis, the spatiotemporal distribution characteristics of ginsenosides, and factors influencing ginsenosides are largely unknown, which make it challenging to enhance the quality and chemical extraction processes of the cultivated American ginseng. This review presents an overview of the pharmacological effects, biosynthesis and spatiotemporal distribution of ginsenosides, with emphasis on the impacts of biotic and abiotic factors on ginsenosides in American ginseng. Modern pharmacological studies have demonstrated that American ginseng has neuroprotective, cardioprotective, antitumor, antidiabetic, and anti-obesity effects. Additionally, most genes involved in the upregulation of ginsenoside biosynthesis have been identified, while downstream regulators (OSCs, CYP450, and UGTs) require further investigation. Futhermore, limited knowledge exists regarding the molecular mechanisms of the impact of biotic and abiotic factors on ginsenosides. Notably, the nonmedicinal parts of American ginseng, particularly its flowers, fibrous roots, and leaves, exhibit higher ginsenoside content than its main roots and account for a considerable amount of weight in the whole plant, representing promising resources for ginsenosides. Herein, the prospects of molecular breeding and metabolic engineering based on multi-omics to improve the unstable quality of cultivated American ginseng and the shortage of ginsenosides are proposed. This review highlights the gaps in the current research on American ginseng and proposes solutions to address these limitations, providing a guide for future investigations into American ginseng ginsenosides.

Cannabis sativa: origin and history, glandular trichome development, and cannabinoid biosynthesis.

Is Cannabis a boon or bane? Cannabis sativa has long been a versatile crop for fiber extraction (industrial hemp), traditional Chinese medicine (hemp seeds), and recreational drugs (marijuana). Cannabis faced global prohibition in the twentieth century because of the psychoactive properties of ∆9-tetrahydrocannabinol; however, recently, the perspective has changed with the recognition of additional therapeutic values, particularly the pharmacological potential of cannabidiol. A comprehensive understanding of the underlying mechanism of cannabinoid biosynthesis is necessary to cultivate and promote globally the medicinal application of Cannabis resources. Here, we comprehensively review the historical usage of Cannabis, biosynthesis of trichome-specific cannabinoids, regulatory network of trichome development, and synthetic biology of cannabinoids. This review provides valuable insights into the efficient biosynthesis and green production of cannabinoids, and the development and utilization of novel Cannabis varieties.

Functional divergence of CYP76AKs shapes the chemodiversity of abietane-type diterpenoids in genus Salvia.

The genus Salvia L. (Lamiaceae) comprises myriad distinct medicinal herbs, with terpenoids as one of their major active chemical groups. Abietane-type diterpenoids (ATDs), such as tanshinones and carnosic acids, are specific to Salvia and exhibit taxonomic chemical diversity among lineages. To elucidate how ATD chemical diversity evolved, we carried out large-scale metabolic and phylogenetic analyses of 71 Salvia species, combined with enzyme function, ancestral sequence and chemical trait reconstruction, and comparative genomics experiments. This integrated approach showed that the lineage-wide ATD diversities in Salvia were induced by differences in the oxidation of the terpenoid skeleton at C-20, which was caused by the functional divergence of the cytochrome P450 subfamily CYP76AK. These findings present a unique pattern of chemical diversity in plants that was shaped by the loss of enzyme activity and associated catalytic pathways.

Natural products of medicinal plants: biosynthesis and bioengineering in post-genomic era.

Globally, medicinal plant natural products (PNPs) are a major source of substances used in traditional and modern medicine. As we human race face the tremendous public health challenge posed by emerging infectious diseases, antibiotic resistance and surging drug prices etc., harnessing the healing power of medicinal plants gifted from mother nature is more urgent than ever in helping us survive future challenge in a sustainable way. PNP research efforts in the pre-genomic era focus on discovering bioactive molecules with pharmaceutical activities, and identifying individual genes responsible for biosynthesis. Critically, systemic biological, multi- and inter-disciplinary approaches integrating and interrogating all accessible data from genomics, metabolomics, structural biology, and chemical informatics are necessary to accelerate the full characterization of biosynthetic and regulatory circuitry for producing PNPs in medicinal plants. In this review, we attempt to provide a brief update on the current research of PNPs in medicinal plants by focusing on how different state-of-the-art biotechnologies facilitate their discovery, the molecular basis of their biosynthesis, as well as synthetic biology. Finally, we humbly provide a foresight of the research trend for understanding the biology of medicinal plants in the coming decades.

Advances in pharmacology, biosynthesis, and metabolic engineering of Scutellaria-specialized metabolites.

Scutellaria Linn., which belongs to the family Lamiaceae, is a commonly used medicinal plant for heat clearing and detoxification. In particular, the roots of S. baicalensis and the entire herb of S. barbata have been widely used in traditional medicine for thousands of years. The main active components of Scutellaria, including: baicalein, wogonin, norwogonin, scutellarein, and their glycosides have potential or existing drug usage. However, the wild resources of Scutellaria plants have been overexploited, and degenerated germplasm resources cannot fulfill the requirements of chemical extraction and clinical usage. Metabolic engineering and green production via microorganisms provide alternative strategies for greater efficiency in the production of natural products. Here, we review the progress of: pharmacological investigations, multi-omics, biosynthetic pathways, and metabolic engineering of various Scutellaria species and their active compounds. In addition, based on multi-omics data, we systematically analyze the phylogenetic relationships of Scutellaria and predict candidate transcription factors related to the regulation of active flavonoids. Finally, we propose the prospects of directed evolution of core enzymes and genome-assisted breeding to alleviate the shortage of plant resources of Scutellaria. This review provides important insights into the sustainable utilization and development of Scutellaria resources.

Comparative Analysis of Chloroplast Genome and New Insights Into Phylogenetic Relationships of Polygonatum and Tribe Polygonateae.

Members of Polygonatum are perennial herbs that have been widely used in traditional Chinese medicine to invigorate Qi, moisten the lung, and benefit the kidney and spleen among patients. However, the phylogenetic relationships and intrageneric taxonomy within Polygonatum have long been controversial because of the complexity of their morphological variations and lack of high-resolution molecular markers. The chloroplast (cp) genome is an optimal model for deciphering phylogenetic relationships in related families. In the present study, the complete cp genome of 26 species of Trib. Polygonateae were de novo assembled and characterized; all species exhibited a conserved quadripartite structure, that is, two inverted repeats (IR) containing most of the ribosomal RNA genes, and two unique regions, large single sequence (LSC) and small single sequence (SSC). A total of 8 highly variable regions (rps16-trnQ-UUG, trnS-GCU-trnG-UCC, rpl32-trnL-UAG, matK-rps16, petA-psbJ, trnT-UGU-trnL-UAA, accD-psaI, and trnC-GCA-petN) that might be useful as potential molecular markers for identifying Polygonatum species were identified. The molecular clock analysis results showed that the divergence time of Polygonatum might occur at ∼14.71 Ma, and the verticillate leaf might be the ancestral state of this genus. Moreover, phylogenetic analysis based on 88 cp genomes strongly supported the monophyly of Polygonatum. The phylogenetic analysis also suggested that Heteropolygonatum may be the sister group of the Polygonatum, but the Disporopsis, Maianthemum, and Disporum may have diverged earlier. This study provides valuable information for further species identification, evolution, and phylogenetic research of Polygonatum.

Deletion and tandem duplications of biosynthetic genes drive the diversity of triterpenoids in Aralia elata.

Araliaceae species produce various classes of triterpene and triterpenoid saponins, such as the oleanane-type triterpenoids in Aralia species and dammarane-type saponins in Panax, valued for their medicinal properties. The lack of genome sequences of Panax relatives has hindered mechanistic insight into the divergence of triterpene saponins in Araliaceae. Here, we report a chromosome-level genome of Aralia elata with a total length of 1.05 Gb. The loss of 12 exons in the dammarenediol synthase (DDS)-encoding gene in A. elata after divergence from Panax might have caused the lack of dammarane-type saponin production, and a complementation assay shows that overexpression of the PgDDS gene from Panax ginseng in callus of A. elata recovers the accumulation of dammarane-type saponins. Tandem duplication events of triterpene biosynthetic genes are common in the A. elata genome, especially for AeCYP72As, AeCSLMs, and AeUGT73s, which function as tailoring enzymes of oleanane-type saponins and aralosides. More than 13 aralosides are de novo synthesized in Saccharomyces cerevisiae by overexpression of these genes in combination. This study sheds light on the diversity of saponins biosynthetic pathway in Araliaceae and will facilitate heterologous bioproduction of aralosides.

Effective prediction of biosynthetic pathway genes involved in bioactive polyphyllins in Paris polyphylla.

The genes in polyphyllins pathway mixed with other steroid biosynthetic genes form an extremely complex biosynthetic network in Paris polyphylla with a giant genome. The lack of genomic data and tissue specificity causes the study of the biosynthetic pathway notably difficult. Here, we report an effective method for the prediction of key genes of polyphyllin biosynthesis. Full-length transcriptome from eight different organs via hybrid sequencing of next generation sequencingand third generation sequencing platforms annotated two 2,3-oxidosqualene cyclases (OSCs), 216 cytochrome P450s (CYPs), and 199 UDP glycosyltransferases (UGTs). Combining metabolic differences, gene-weighted co-expression network analysis, and phylogenetic trees, the candidate ranges of OSC, CYP, and UGT genes were further narrowed down to 2, 15, and 24, respectively. Beside the three previously characterized CYPs, we identified the OSC involved in the synthesis of cycloartenol and the UGT (PpUGT73CR1) at the C-3 position of diosgenin and pennogenin in P. polyphylla. This study provides an idea for the investigation of gene cluster deficiency biosynthesis pathways in medicinal plants.

Comparative and phylogenetic analyses of the chloroplast genomes of species of Paeoniaceae.

Plants belonging to family Paeoniaceae are not only economically important ornamental plants but also medicinal plants used as an important source of traditional Chinese medicine. Owing to the complex network evolution and polyploidy evolution of this family, its systematics and taxonomy are controversial and require a detailed investigation. In this study, three complete chloroplast genomes of sect. Paeonia, one of the sections of Paeonia, were sequenced and then analysed together with 16 other published chloroplast genomes of Paeoniaceae species. The total lengths of the chloroplast genomes of these species were 152,153-154,405 bp. A total of 82-87 protein-coding genes, 31-40 tRNA genes and 8 rRNA genes were annotated. Bioinformatics analysis revealed 61-74 simple sequence repeats (SSRs) in the chloroplast genomes, most of which have A/T base preference. Codon usage analysis showed that A/U-ending codons were more positive than C/G-ending codons, and a slight bias in codon usage was observed in these species. A comparative analysis of these 19 species of Paeoniaceae was then conducted. Fourteen highly variable regions were selected for species relationship study. Phylogenetic analysis revealed that the species of sect. Paeonia gathered in one branch and then divided into different small branches. P. lactiflora, P. anomala, P. anomala subsp. veitchii and P. mairei clustered together. P. intermedia was related to P. obovata and P. obovata subsp. willmottiae. P. emodi was the sister to all other species in the sect. Paeonia.

Identification and phylogenetic analysis of five Crataegus species (Rosaceae) based on complete chloroplast genomes.

MAIN CONCLUSION: The chloroplast genomes of the five Crataegus species were shown to have a conserved genome structure. Complete chloroplast genome sequences were more suitable than highly variable regions for the identification and phylogenetic analysis of Crataegus species. Hawthorn, which is commonly used as a traditional Chinese medicine, is one of the most popular sour fruits and has high economic value. Crataegus pinnatifida var. pinnatifida and C. pinnatifida var. major are frequently adulterated with other Crataegus species on the herbal medicine market. However, most Crataegus plants are difficult to identify using traditional morphological methods. Here, we compared five Crataegus chloroplast (CP) genomes comprising two newly sequenced (i.e., C. pinnatifida var. pinnatifida and C. pinnatifida var. major) and three previously published CP genomes. The CP genomes of the five Crataegus species had a conserved genome structure, gene content and codon usage. The total length of the CP genomes was 159,654-159,865 bp. A total of 129-130 genes, including 84-85 protein-coding genes, 37 tRNA genes and 8 rRNA genes, were annotated. Bioinformatics analysis revealed 96-103 simple sequence repeats (SSRs) and 48-70 long repeats in the five CP genomes. Combining the results of mVISTA and nucleotide diversity, five highly variable regions were screened for species identification and relationship studies. Maximum likelihood trees were constructed on the basis of complete CP genome sequences and highly variable regions. The results showed that the former had higher discriminatory power for Crataegus species, indicating that the complete CP genome could be used as a super-barcode to accurately authenticate the five Crataegus species.

Comparative Genome Analysis of Scutellaria baicalensis and Scutellaria barbata Reveals the Evolution of Active Flavonoid Biosynthesis.

Scutellaria baicalensis (S. baicalensis) and Scutellaria barbata (S. barbata) are common medicinal plants of the Lamiaceae family. Both produce specific flavonoid compounds, including baicalein, scutellarein, norwogonin, and wogonin, as well as their glycosides, which exhibit antioxidant and antitumor activities. Here, we report chromosome-level genome assemblies of S. baicalensis and S. barbata with quantitative chromosomal variation (2n = 18 and 2n = 26, respectively). The divergence of S. baicalensis and S. barbata occurred far earlier than previously reported, and a whole-genome duplication (WGD) event was identified. The insertion of long terminal repeat elements after speciation might be responsible for the observed chromosomal expansion and rearrangement. Comparative genome analysis of the congeneric species revealed the species-specific evolution of chrysin and apigenin biosynthetic genes, such as the S. baicalensis-specific tandem duplication of genes encoding phenylalanine ammonia lyase and chalcone synthase, and the S. barbata-specific duplication of genes encoding 4-CoA ligase. In addition, the paralogous duplication, colinearity, and expression diversity of CYP82D subfamily members revealed the functional divergence of genes encoding flavone hydroxylase between S. baicalensis and S. barbata. Analyzing these Scutellaria genomes reveals the common and species-specific evolution of flavone biosynthetic genes. Thus, these findings would facilitate the development of molecular breeding and studies of biosynthesis and regulation of bioactive compounds.

Aloe extract inhibits porcine epidemic diarrhea virus in vitro and in vivo.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhoea and high mortality in neonatal suckling piglets, leading to significant economic losses to the swine industry. Currently there are no adequate control strategies against circulating PEDV variants, making an urgent need to exploit effect antiviral therapies to compensate for vaccines. Here, we report that Aloe extract can hamper completely the proliferation of PEDV at a non-cytotoxic concentration of 16 mg/mL determined by CCK-8 assay in Vero and IPEC-J2 cells in vitro. Furthermore, time course analysis indicated the extract exerted its inhibition at the late stage of the viral life cycle. Moreover, we also confirmed that the extract can inactivated PEDV directly but did not act on the viral genome and S1 protein. Importantly, the extract at a relatively safety concentration of 100 mg/kg of body weight, which was confirmed in mice, could reduce virus load and pathological change in intestinal tract of pigs and protect newborn piglets from lethal challenge with highly pathogenic PEDV variant GDS01 infection, indicating that Aloe extract efficiently inhibited PEDV infection in vivo. Collectively, our findings suggest that the aqueous extract from the Aloe could inhibit PEDV replication in vitro and in vivo and might be a good target for drug development against PEDV.

Biosynthesis of selenoproteins by Saccharomyces cerevisiae and characterization of its antioxidant activities.

The study investigated biosynthesis of selenoproteins by Saccharomyces. cerevisiae using inorganic selenium. Selenium supplement via two stages was carried out during fermentation and the physicochemical characteristics of selenoproteins and its antioxidant activities were examined through in vitro assessment procedures. After fermentation, dry cells weight (7.47 g/L) and selenium content (3079.60 µg/kg) in the yeast were achieved when fermentation time points at the 6th hour and the 9th hour were chosen to supplement 30% and 70% of 30 µg/mL Na2SeO3 respectively. A maximal yield of selenium content in selenoproteins reached 1013.07 µg/g under optimized culture conditions and was 133-fold higher than the control. One new band with molecular weight of 26.76 KDa appeared in conjugated selenoproteins of sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Surface structure of selenoproteins and the control was different by Scanning electron microscopy images. Infrared spectrometry analysis demonstrated that groups of HSe, SeO and C-Se-O involved in selenoproteins were important pieces of evidence showing presence of Se embedded in the protein molecule. Selenoproteins showed strong antioxidant activities on DPPH·, OH and ·O2-, which was much higher than the control proteins. Therefore, the study provided an efficient selenium-enriched culture method of inorganic selenite to organic selenium and basis for selenoproteins applications.

Genome-wide identification and analysis of AP2/ERF transcription factors related to camptothecin biosynthesis in Camptotheca acuminata.

Camptotheca acuminata produces camptothecin (CPT), a monoterpene indole alkaloid (MIA) that is widely used in the treatment of lung, colorectal, cervical, and ovarian cancers. Its biosynthesis pathway has attracted significant attention, but the regulation of CPT biosynthesis by the APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors (TFs) remains unclear. In this study, a systematic analysis of the AP2/ERF TFs family in C. acuminata was performed, including phylogeny, gene structure, conserved motifs, and gene expression profiles in different tissues and organs (immature bark, cotyledons, young flower, immature fruit, mature fruit, mature leaf, roots, upper stem, and lower stem) of C. acuminata. A total of 198 AP2/ERF genes were identified and divided into five relatively conserved subfamilies, including AP2 (26 genes), DREB (61 genes), ERF (92 genes), RAV (18 genes), and Soloist (one gene). The combination of gene expression patterns in different C. acuminata tissues and organs, the phylogenetic tree, the co-expression analysis with biosynthetic genes, and the analysis of promoter sequences of key enzymes genes involved in CPT biosynthesis pathways revealed that eight AP2/ERF TFs in C. acuminata might be involved in CPT synthesis regulation, which exhibit relatively high expression levels in the upper stem or immature bark. Among these, four genes (CacAP2/ERF123, CacAP2/ERF125, CacAP2/ERF126, and CacAP2/ERF127) belong to the ERF-B2 subgroup; two genes (CacAP2/ERF149 and CacAP2/ERF152) belong to the ERF-B3 subgroup; and two more genes (CacAP2/ERF095 and CacAP2/ERF096) belong to the DREB-A6 subgroup. These results provide a foundation for future functional characterization of the AP2/ERF genes to enhance the biosynthesis of CPT compounds of C. acuminata.

Genome-wide identification of abscisic acid (ABA) receptor pyrabactin resistance 1-like protein (PYL) family members and expression analysis of PYL genes in response to different concentrations of ABA stress in Glycyrrhiza uralensis.

As abscisic acid (ABA) receptor, the pyrabactin resistance 1-like (PYR/PYL) protein (named PYL for simplicity) plays an important part to unveil the signal transduction of ABA and its regulatory mechanisms. Glycyrrhiza uralensis, a drought-tolerant medicinal plant, is a good model for the mechanism analysis of ABA response and active compound biosynthesis. However, knowledge about PYL family in G. uralensis remains largely unknown. Here, 10 PYLs were identified in G. uralensis genome. Characterization analysis indicated that PYLs in G. uralensis (GuPYLs) are relatively conserved. Phylogenetic analysis showed that GuPYL1-3 belongs to subfamily I, GuPYL4-6 and GuPYL10 belong to subfamily II and GuPYL7-9 belongs to subfamily III. In addition, transcriptome data presented various expression levels of GuPYLs under different exogenous ABA stresses. The expression pattern of GuPYLs was verified by Quantitative real-time polymerase chain reaction (qRT-PCR). The study proved that GuPYL4, GuPYL5, GuPYL8 and GuPYL9 genes are significantly up-regulated by ABA stress and the response process is dynamic. This study paves the way for elucidating the regulation mechanism of ABA signal to secondary metabolites and improving the cultivation and quality of G. uralensis using agricultural strategies.

Identification of dibucaine derivatives as novel potent enterovirus 2C helicase inhibitors: In vitro, in vivo, and combination therapy study.

Enterovirus A71 (EV-A71) is a human pathogen causing hand, foot and mouth disease (HFMD) which seriously threatened the safety and lives of infants and young children. However, there are no licensed direct antiviral agents to cure the HFMD. In this study, a series of quinoline formamide analogues as effective enterovirus inhibitors were developed, subsequent systematic structure-activity relationship (SAR) studies demonstrated that these quinoline formamide analogues exhibited good potency to treat EV-A71 infection. As described, the most efficient EV-A71 inhibitor 6i showed good anti-EV-A71 activity (EC50 = 1.238 µM) in RD cells. Furthermore, compound 6i could effectively prevent death of virus infected mice at dose of 6 mg/kg. When combined with emetine (0.1 mg/kg), this treatment could completely prevent the clinical symptoms and death of virus infected mice. Mechanism study indicated that compound 6i inhibited EV-A71 via targeting 2C helicase, thus impeding RNA remodeling and metabolism. Taken together, these data indicated that 6i is a promising EV-A71 inhibitor and worth extensive preclinical investigation as a lead compound.