Genetic diversities in wild and cultivated populations of the two closely-related medical plants species, Tripterygium Wilfordii and T. Hypoglaucum (Celastraceae).

BACKGROUND: The sustainable supply of medicinal plants is important, and cultivating and domesticating them has been suggested as an optimal strategy. However, this can lead to a loss of genetic diversity. Tripterygium wilfordii Hook. f. is a medicinal plant commonly used in traditional Chinese medicine, but its wild populations are dwindling due to excessive harvesting. To protect the species and meet the increasing demand, it is urgent to cultivate it on a large scale. However, distinguishing between T. wilfordii and T. hypoglaucum, two similar species with different medicinal properties, is challenging. Therefore, it is crucial to understand the genetic diversity and population structure of these species for their sustainable utilization. RESULTS: In this study, we investigated the genetic diversity and population structure of the two traditional medicinal semiwoody vines plant species, Tripterygium wilfordii and T. hypoglaucum, including wild and cultivated populations using chloroplast DNA (cpDNA) sequences and microsatellite loci. Our results indicated that the two species maintain a high level of genetic divergence, indicating possible genetic bases for the different contents of bioactive compounds of the two species. T. wilfordii showed lower genetic diversity and less subdivided population structures of both markers than T. hypoglaucum. The potential factors in shaping these interesting differences might be differentiated pollen-to-seed migration rates, interbreeding, and history of population divergence. Analyses of cpDNA and microsatellite loci supported that the two species are genetically distinct entities. In addition, a significant reduction of genetic diversity was observed for cultivated populations of the two species, which mainly resulted from the small initial population size and propagated vegetative practice during their cultivation. CONCLUSION: Our findings indicate significant genetic divergence between T. wilfordii and T. hypoglaucum. The genetic diversity and population structure analyses provide important insights into the sustainable cultivation and utilization of these medicinal plants. Accurate identification and conservation efforts are necessary for both species to ensure the safety and effectiveness of crude drug use. Our study also highlighted the importance of combined analyses of different DNA markers in addressing population genetics of medicinal plants because of the contrasts of inheritance and rates of gene flow. Large-scale cultivation programs should consider preserving genetic diversity to enhance the long-term sustainability of T. wilfordii and T. hypoglaucum. Our study proposed that some populations showed higher genetic diversity and distinctness, which can be considered with priority for conservation and as the sources for future breeding and genetic improvement.

Moxibustion prevents tripterygium glycoside-induced oligoasthenoteratozoospermia in rats via reduced oxidative stress and modulation of the Nrf2/HO-1 signaling pathway.

Oligoasthenoteratozoospermia (OAT) decreases male fertility, seriously affecting the production of offspring. This study clarified the preventive impact of different moxibustion frequencies on OAT and selected the optimal frequency to elucidate the underlying mechanism. An OAT rat model was constructed by gavage of tripterygium glycosides (TGS) suspension. Daily moxibustion (DM) or alternate-day moxibustion (ADM) was administered on the day of TGS suspension administration. Finally, we selected DM for further study based on sperm quality and DNA fragmentation index, testicular and epididymal morphology, and reproductive hormone level results. Subsequently, the oxidative stress (OS) status was evaluated by observing the OS indices levels; malondialdehyde (MDA), 8-hydroxy-deoxyguanosine (8-OHdG), total antioxidant capacity (T-AOC), and total superoxide dismutase (T-SOD) in testicular tissue using colorimetry and enzyme-linked immunosorbent assay. Furthermore, heme oxygenase 1 (HO-1) and nuclear factor erythropoietin-2-related factor 2 (Nrf2) were evaluated using Western blotting. Immunohistochemistry was employed to locate and assess the expression of HO-1 and Nrf2 protein, while quantitative real-time polymerase chain reaction was utilized to detect their mRNA expression. MDA and 8-OHdG levels decreased following DM treatment, while T-SOD and T-AOC increased, suggesting that DM may prevent TGS-induced OAT in rats by decreasing OS in the testis. Furthermore, protein and mRNA expression of Nrf2 and HO-1 in the testis were elevated, indicating that DM may reduce OS by activating the signaling pathway of Nrf2/HO-1. Therefore, DM could prevent OAT in rats via the Nrf2/HO-1 pathway, thereby presenting a promising therapeutic approach against OAT.

Characterization of the Cytochrome P450 CYP716C52 in Celastrol Biosynthesis and Its Applications in Engineered Saccharomyces cerevisiae.

Celastrol is a bioactive pentacyclic triterpenoid with promising therapeutic effects that is mainly distributed in Celastraceae plants. Although some enzymes involved in the celastrol biosynthesis pathway have been reported, many biosynthetic steps remain unknown. Herein, transcriptomics and metabolic profiles of multiple species in Celastraceae were integrated to screen for cytochrome P450s (CYPs) that are closely related to celastrol biosynthesis. The CYP716 enzyme, TwCYP716C52, was found to be able to oxidize the C-2 position of polpunonic acid, a precursor of celastrol, to form the wilforic acid C. RNAi-mediated repression of TwCYP716C52 in Tripterygium wilfordii suspension cells further confirmed its involvement in celastrol biosynthesis. The C-2 catalytic mechanisms of TwCYP716C52 were further explored by using molecular docking and site-directed mutagenesis experiments. Moreover, a modular optimization strategy was used to construct an engineered yeast to produce wilforic acid C at a titer of 5.8 mg·L-1. This study elucidates the celastrol biosynthetic pathway and provides important functional genes and sufficient precursors for further enzyme discovery.

CYP72D19 from Tripterygium wilfordii catalyzes C-2 hydroxylation of abietane-type diterpenoids.

KEY MESSAGE: CYP72D19, the first functional gene of the CYP72D subfamily, catalyzes the C-2 hydroxylation of abietane-type diterpenoids. The abietane-type diterpenoids, e.g., triptolide, tripdiolide, and 2-epitripdiolide, are the main natural products for the anti-tumor, anti-inflammatory, and immunosuppressive activities of Tripterygium wilfordii, while their biosynthetic pathways are not resolved. Here, we cloned and characterized the CYP72D19-catalyzed C-2 hydroxylation of dehydroabietic acid, a compound that has been proven to be a biosynthetic intermediate in triptolide biosynthesis. Through molecular docking and site-directed mutagenesis, L386, L387, and I493 near the active pocket were found to have an important effect on the enzyme activity, which also indicates that steric hindrance of residues plays an important role in function. In addition, CYP72D19 also catalyzed a variety of abietane-type diterpenoids with benzene ring, presumably because the benzene ring of the substrate molecule stabilized the C-ring, allowing the protein and the substrate to form a relatively stable spatial structure. This is the first demonstration of CYP72D subfamily gene function. Our research provides important genetic elements for the structural modification of active ingredients and the heterologous production of other 2-hydroxyl abietane-type natural products.

Protective effects of Tripterygium glycoside on IL-1ß-induced inflammation and apoptosis of rat chondrocytes via microRNA-216a-5p/TLR4/NF-κB axis.

BACKGROUND: This study is designed to fill the research gap concerning the efficacy of Tripterygium glycoside (TG) on Interleukin-1ß (IL-1ß)-induced inflammation and injury in chondrocytes. METHODS: Chondrocytes were isolated from Sprague-Dawley rats. After the treatment with IL-1ß and TG and transfection, the viability and apoptosis of chondrocytes were determined via Cell Counting Kit-8 (CCK-8) assay and flow cytometry. The levels of inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-8 were determined by enzyme-linked immunosorbent assay (ELISA). Relative expression levels of potential microRNAs (miRNAs, miRs) that may target toll-like receptor 4 (TLR4), as well as apoptosis- and TLR4/nuclear factor-κB (TLR4/NF-κB) pathway-associated factors were quantified using quantitative real-time (qRT) PCR and western blot. The targeting relationship between miR-216a-5p and TLR4 was predicted by TargetScan and further confirmed by dual-luciferase reporter assay. RESULTS: The viability was reduced yet the apoptosis and inflammation were promoted in IL-1ß-treated chondrocytes, where upregulation of Bax, Cleaved caspase 3, TLR4, Myeloid differentiation factor 88 (MyD88), phosphorylation of P65 and IκBα yet downregulation of Bcl-2 and IκBα were evidenced. Strikingly, the above changes were reversed by TG. TG also offset the effects of IL-1ß on repressing the expression of miR-216a-5p, the miRNA targeting TLR4. Additionally, TLR4 overexpression neutralized the impacts of TG upon viability, apoptosis, and TLR4 expression in IL-1ß-treated chondrocytes, while all these effects induced by TLR4 overexpression could be restored by miR-216a-5p. CONCLUSIONS: TG protects chondrocytes against IL-1ß-induced inflammation and apoptosis via miR-216a-5p/TLR4/NF-κB axis.

Tripterygium wilfordii cytochrome P450s catalyze the methyl shift and epoxidations in the biosynthesis of triptonide.

The diterpenoid triepoxides triptolide and triptonide from Tripterygium wilfordii (thunder god wine) exhibit unique bioactivities with potential uses in disease treatment and as a non-hormonal male contraceptives. Here, we show that cytochrome P450s (CYPs) from the CYP71BE subfamily catalyze an unprecedented 18(4→3) methyl shift required for biosynthesis of the abeo-abietane core structure present in diterpenoid triepoxides and in several other plant diterpenoids. In combination with two CYPs of the CYP82D subfamily, four CYPs from T. wilfordii are shown to constitute the minimal set of biosynthetic genes that enables triptonide biosynthesis using Nicotiana benthamiana and Saccharomyces cerevisiae as heterologous hosts. In addition, co-expression of a specific T. wilfordii cytochrome b5 (Twcytb5-A) increases triptonide output more than 9-fold in S. cerevisiae and affords isolation and structure elucidation by NMR spectroscopic analyses of 18 diterpenoids, providing insights into the biosynthesis of diterpenoid triepoxides. Our findings pave the way for diterpenoid triepoxide production via fermentation.

Probing the functions of friedelane-type triterpene cyclases from four celastrol-producing plants.

Triterpenes are among the most diverse plant natural products, and their diversity is closely related to various triterpene skeletons catalyzed by different 2,3-oxidosqualene cyclases (OSCs). Celastrol, a friedelane-type triterpene with significant bioactivities, is specifically distributed in higher plants, such as Celastraceae species. Friedelin is an important precursor for the biosynthesis of celastrol, and it is synthesized through the cyclization of 2,3-oxidosqualene, with the highest number of rearrangements being catalyzed by friedelane-type triterpene cyclases. However, the molecular mechanisms underlying the catalysis of friedelin production by friedelane-type triterpene cyclases have not yet been fully elucidated. In this study, transcriptome data of four celastrol-producing plants from Celastraceae were used to identify a total of 21 putative OSCs. Through functional characterization, the friedelane-type triterpene cyclases were separately verified in the four plants. Analysis of the selection pressure showed that purifying selection acted on these OSCs, and the friedelane-type triterpene cyclases may undergo weaker selective restriction during evolution. Molecular docking and site-directed mutagenesis revealed that changes in some amino acids that are unique to friedelane-type triterpene cyclases may lead to variations in catalytic specificity or efficiency, thereby affecting the synthesis of friedelin. Our research explored the functional diversity of triterpene synthases from a multispecies perspective. It also provides some references for further research on the relative mechanisms of friedelin biosynthesis.

Functional characterization and substrate promiscuity of sesquiterpene synthases from Tripterygium wilfordii.

Acyclic terpenes, commonly found in plants, are of high physiological importance and commercial value, and their diversity was controlled by different terpene synthases. During the screen of sesquiterpene synthases from Tripterygium wilfordii, we observed that Ses-TwTPS1-1 and Ses-TwTPS2 promiscuously accepted GPP, FPP, and GGPP to produce corresponding terpene alcohols (linalool/nerolidol/geranyllinalool). The Ses-TwTPS1-2, Ses-TwTPS3, and Ses-TwTPS4 also showed unusual substrate promiscuity by catalyzing GGPP or GPP in addition to FPP as substrate. Furthermore, key residues for the generation of diterpene product, (E, E)-geranyllinalool, were screened depending on mutagenesis studies. The functional analysis of Ses-TwTPS1-1:V199I and Ses-TwTPS1-2:I199V showed that Val in 199 site assisted the produce of diterpene product geranyllinalool by enzyme mutation studies, which indicated that subtle differences away from the active site could alter the product outcome. Moreover, an engineered sesquiterpene high-yielding yeast that produced 162 mg/L nerolidol in shake flask conditions was constructed to quickly identify the function of sesquiterpene synthases in vivo and develop potential applications in microbial fermentation. Our functional characterization of acyclic sesquiterpene synthases will give some insights into the substrate promiscuity of diverse acyclic terpene synthases and provide key residues for expanding the product portfolio.

Cytochrome P450 catalyses the 29-carboxyl group formation of celastrol.

Celastrol, a potent anticancer and anti-obesity drug, was first isolated from Tripterygium wilfordii Hook. f. and it is produced in small quantities in many members of the Celastraceae family. The heterologous reconstitution of celastrol biosynthesis could be a promising method for the efficient production of celastrol and natural and unnatural derivatives thereof, yet only part of the biosynthetic pathway is known. Here, we report a cytochrome P450 monooxygenase (TwCYP712K1) from T. wilfordii that performs the three-step oxidation of friedelin to polpunonic acid in the celastrol pathway. Heterologous expression of TwCYP712K1 showed that TwCYP712K1 catalyses not only the transformation of friedelin to polpunonic acid but also the oxidation of ß-amyrin or α-amyrin. The role of TwCYP712K1 in the biosynthesis of celastrol was further revealed via RNA interference. Some key residues of TwCYP712K1 were also screened by molecular docking and site-directed mutagenesis. Our results lay a solid foundation for further elucidating the biosynthesis of celastrol and related triterpenoids.

Identification and functional characterization of a PDR transporter in Tripterygium wilfordii Hook.f. that mediates the efflux of triptolide.

KEY MESSAGE: TwPDR1, a PDR transporter from Tripterygium wilfordii Hook.f., was proved to efflux triptolide and its stability could be enhanced by A1033T mutation. Triptolide, an abietane-type diterpene in Tripterygium wilfordii Hook.f., possesses many pharmacological activities. However, triptolide is in short supply and very expensive because it is present at low amounts in natural plants and lack alternative production methods. Transporter engineering, which increases the extracellular secretion of secondary metabolites in in vitro culture systems, is an effective strategy in metabolic engineering but is rarely reported. In this study, TwPDR1, a pleiotropic drug resistance-type ATP binding cassette transporter, was identified as the best efflux pump candidate for diterpenoids through bioinformatics analysis. TwPDR1 was located in the plasma membrane, highly expressed in adventitious roots, and induced by methyl jasmonate. The triptolide efflux function of TwPDR1 was confirmed by transient expression in tobacco BY-2 cells and by downregulation via RNA interference in the native host. However, the overexpression of TwPDR1 had a limited effect on the secretion of triptolide. As shown by previous studies, a single amino acid mutation might increase the abundance of TwPDR1 by increasing protein stability. We identified the A1033 residue in TwPDR1 by sequence alignment and confirmed that A1033T mutation could increase the expression of TwPDR1 and result in the higher release ratio of triptolide (78.8%) of the mutants than that of control (60.1%). The identification and functional characterization of TwPDR1 will not only provide candidate gene material for the metabolic engineering of triptolide but also guide other transporter engineering researches in the future.

Network Pharmacology to Uncover the Molecular Mechanisms of Action of LeiGongTeng for the Treatment of Nasopharyngeal Carcinoma.

BACKGROUND Nasopharyngeal carcinoma (NPC) is a common head and neck cancer epidemic in southern China and southeast Asia. LeiGongTeng has been widely used for the treatment of cancers. The purpose of this study was to determine the pharmacological mechanism of action of LeiGongTeng in the treatment of NPC using a network pharmacological approach. MATERIAL AND METHODS The traditional Chinese medicine systems pharmacology (TCMSP) database was used to identify active ingredients and associated target proteins for LeiGongTeng. Cytoscape was utilized to create a drug-disease network and topology analysis was conducted to analyze the degree of each ingredient. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) online tool was applied for the construction and analysis of the protein-protein interaction (PPI) network, while Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) functional analyses were utilized to determine drug-disease common genes. RESULTS 22 active ingredients including kaempferol, nobiletin, and beta-sitosterol, and 30 drug-disease common genes including VEGFA, CASP3, ESR1, and RELA were identified. GO analysis indicated that 94 biological processes, including RNA polymerase II, apoptotic process, response to drug, cell adhesion, and response to hypoxia, were found to be associated with NPC. The KEGG enrichment analysis showed that 58 pathways, including the PI3K-Akt signaling pathway, microRNAs in cancer, tumor necrosis factor (TNF) signaling pathway and pathways in cancer were found to be associated with NPC. CONCLUSIONS LeiGongTeng exerts its therapeutic effect through various biological processes and signaling pathways since it acts on several target genes. Systematic pharmacology can be used to predict the underlying function of LeiGongTeng and its mechanism of action in NPC.

Genome of Tripterygium wilfordii and identification of cytochrome P450 involved in triptolide biosynthesis.

Triptolide is a trace natural product of Tripterygium wilfordii. It has antitumor activities, particularly against pancreatic cancer cells. Identification of genes and elucidation of the biosynthetic pathway leading to triptolide are the prerequisite for heterologous bioproduction. Here, we report a reference-grade genome of T. wilfordii with a contig N50 of 4.36 Mb. We show that copy numbers of triptolide biosynthetic pathway genes are impacted by a recent whole-genome triplication event. We further integrate genomic, transcriptomic, and metabolomic data to map a gene-to-metabolite network. This leads to the identification of a cytochrome P450 (CYP728B70) that can catalyze oxidation of a methyl to the acid moiety of dehydroabietic acid in triptolide biosynthesis. We think the genomic resource and the candidate genes reported here set the foundation to fully reveal triptolide biosynthetic pathway and consequently the heterologous bioproduction.

A class I TGA transcription factor from Tripterygium wilfordii Hook.f. modulates the biosynthesis of secondary metabolites in both native and heterologous hosts.

Class I TGA transcription factors (TFs) are known to participate in plant resistance responses, however, their regulatory functions in the biosynthesis of secondary metabolites were rarely revealed. In this study, a class I TGA TF, TwTGA1, from Tripterygium wilfordii Hook.f. was cloned and characterized. Overexpression of TwTGA1 in T. wilfordii Hook.f. cells increased the production of triptolide and two sesquiterpene pyridine alkaloids, which was further enhanced by methyl jasmonate (MeJA) treatment. RNA interference of TwTGA1 showed no significant effects on the production of these metabolites, indicating the existence of other TGA partner(s) with overlapping functions. Heterologous expression of TwTGA1 in tobacco By-2 cells promoted the biosynthesis of pyridine alkaloids. Under the elicitation of MeJA, the contents of nonpyrrolidine alkaloids further increased but not for nicotine. TwTGA1 could induce the expression of Putrescine N-methyltransferase (PMT) and N-methylputrescine oxidase 1 (MPO1) through binding to their promoters. Finally, transient expression of TwTGA1 in leaves of Catharanthus roseus changed both the profiles of vinca alkaloids (increased contents of serpentine and catharanthine, but decreased that of vinblastine) and the expressions of biosynthesis-related genes. The metabolic and transcriptional data indicated a relationship between jasmonic acid signaling pathway and the functions of TwTGA1.

Identification and functional characterization of squalene epoxidases and oxidosqualene cyclases from Tripterygium wilfordii.

KEY MESSAGE: We cloned two squalene epoxidases and five oxidosqualene cyclases, and identified their function using CRISPR/Cas9 tool and yeast heterologous expression. Triterpenes are the main active ingredients of Tripterygium wilfordii Hook.f., a traditional Chinese medicinal plant with many encouraging preclinical applications. However, the biosynthetic pathways of triterpenes in this plant are poorly understood. Here, we report on the isolation and identification of two squalene epoxidases (SQE6 and SQE7) and five oxidosqualene cyclases (OSC4-8) from T. wilfordii. Yeast complementation assays showed that TwSQE6 and TwSQE7 can functionally complement an erg1 yeast mutant that was constructed using the CRISPR/Cas9 system. The putative OSC genes were functionally characterized by heterologous expression in yeast. GC/MS analysis of the fermentation products of the transgenic yeast showed that both TwOSC4 and TwOSC6 are cycloartenol synthases, while TwOSC8 is a ß-amyrin synthase. The discovery of these genes expands our knowledge of key enzymes in triterpenoid biosynthesis, and provides additional target genes for increasing the production of triterpenes in T. wilfordii tissue cultures by disrupting competing pathways, or in chassis cells by reconstituting the triterpenoid biosynthetic pathway.

The expression of TwDXS in the MEP pathway specifically affects the accumulation of triptolide.

1-Deoxy-d-xylulose-5-phosphate synthase (DXS) is the first enzyme in the plant 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of terpenoid synthesis. TwDXS is a prominent protein in the Tripterygium wilfordii proteome, with especially high expression in the root periderm. It is significantly regulated by methyl jasmonate. Here, we studied the influence of TwDXS expression on bioactive terpenoids in T. wilfordii. Specific fragments of TwDXS (GenBank: AKP20998.1) with lengths of 2148 and 437 bp were amplified to construct the overexpression (OE) and RNA-interference (RNAi) vectors, respectively. After transformation of suspension cells, the expression of TwDXS and genes related to the terpenoid biosynthetic pathway was measured using qRT-PCR. TwDXS mRNA level was 153 and 43% of the control in the OE and RNAi lines. Related genes in the 2-C-methyl-d-erythritol 4-phosphate (MEP), mevalonic acid (MVA) and downstream pathways showed similar trends to the changes of TwDXS expression. Ultra Performance Liquid Chromatography (UPLC) was employed to measure the accumulation of terpenoids. Importantly, the triptolide content showed significant differences in both the TwDXS OE (222.35% of the control) and RNAi (34.86% of the control). However, there were no obvious changes in the celastrol content. In this study, we verified that the expression of TwDXS affects triptolide but not celastrol in T. wilfordii via both TwDXS OE and RNAi experiments.

[Cloning and characterization of a monoterpene synthase gene from Tripterygium wilfordii].

Tripterygium wilfordii is a medicinal plant commonly used in the treatment of rheumatoid arthritis,and with pharmacological activities in anti-tumor and obesity treatment. The known active ingredients in T. wilfordii are mainly terpenoids,but with very low content. Therefore,the analysis of the biosynthesis pathway of terpenoids in T. wilfordii has become a research hotspot to solve the problem of its resources. Terpenoid synthase( TPS) is a key enzyme that catalyzes the formation of a wide variety of terpenoid skeletons. In this study,a gene fragment with an ORF of 1 785 bp was cloned from T. wilfordii. Bioinformatics analysis was performed using NCBI's BLASTP,ProtParam and Interpro online tools and MEGA 6.0 software. The response of this gene to methyl jasmonate was also detected by real-time fluorescent quantitative PCR,and its catalytic function was verified by prokaryotic expression and in vitro enzymatic assay. Bioinformatics analysis indicated that the amino acid sequence encoded by this gene had both N-terminal domain and C-terminal domain of TPS,as well as the DDxx D conserved domain of the class I of TPS family. And Tw MTS gathered together with TPS-b subfamily in the Neighbor-Joining Tree constructed with known homologous TPSs. The results of RT-PCR showed that 50 µmol·L-1 MeJA 12 h could increase the expression of Tw MTS to 735 times in the control group at 12 h,and 1 644 times at 24 h. In addition,in vitro enzymatic reaction results showed that Tw MTS can catalyze the production of ß-citronellol with GPP as substrate,indicating that Tw MTS was a monoterpene synthase. The above results provided a new element for the synthetic biology database of T. wilfordii terpenoids,and laid the foundation for future biosynthesis research.

Differential expression of the TwHMGS gene and its effect on triptolide biosynthesis in Tripterygium wilfordii.

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the pH7WG2D and pK7GWIWG2D vectors to respectively overexpress and silence, TwHMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to 70% in the expression level compared to cells with empty vector controls. During TwHMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7v2 was significantly upregulated to the control. In the RNAi group, the expression of TwHMGR, TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of TwHMGS determined the production of triptolide in T. wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-CoA was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA.

The gibberellin 13-oxidase that specifically converts gibberellin A9 to A20 in Tripterygium wilfordii is a 2-oxoglutarate-dependent dioxygenase.

MAIN CONCLUSION: A novel GA13-oxidase ofTripterygium wilfordii, TwGA13ox, is a 2-oxoglutarate-dependent dioxygenase. It specifically catalyzes the conversion of GA9to GA20, but not GA4to GA1. Gibberellins (GAs) play essential roles in plant growth and development. Previous characterization of GA20- and GA3-oxidases yielded a large number of genetic elements that can interconvert different GAs. However, enzymes that catalyze the 13-hydroxylation step are rarely identified. Here, we report that the GA13-oxidase of Tripterygium wilfordii, TwGA13ox, is a 2-oxoglutarate-dependent dioxygenase instead of reported cytochrome P450 oxygenases, among 376 differential proteins in comparative proteomics. Phylogenetic analysis showed that the enzyme resides in its own independent branch in the DOXC class. Unexpectedly, it specifically catalyzes the conversion of GA9 to GA20, but not GA4 to GA1. Contrary to the previous research, TwGA13ox transcriptional expression was upregulated ~ 146 times by exogenous application of methyl jasmonate (MeJA). RNAi targeting of TwGA13ox in T. wilfordii led to an 89.9% decrease of triptolide, a diterpenoid epoxide with extensive anti-inflammatory and anti-tumor properties. In subsequent MeJA supplementation experiments, triptolide production increased 13.4-times. TwGA13ox displayed root-specific expression. Our results provide a new GA13-oxidase from plants and elucidate the metabolic associations within the diterpenoid biosynthetic pathway (GAs, triptolide) at the genetic level.

Analysis of the role of geranylgeranyl diphosphate synthase 8 from Tripterygium wilfordii in diterpenoids biosynthesis.

Tripterygium wilfordii is known to contain various types of bioactive diterpenoids that exhibit many remarkable activities. Many studies have recently been targeted toward the elucidation of the diterpenoids biosynthetic pathways in attempts to obtain these compounds with a view to solving the dilemma of low yield in plants. However, the short-chain prenyltransferases (SC-PTSs) responsible for the formation of geranylgeranyl diphosphate (GGPP), a crucial precursor for synthesizing the skeleton structures of diterpenoids, have not been characterized in depth. Here, T. wilfordii transcriptome data were used to identify eight putative GGPPSs, including two small subunits of geranyl diphosphate synthase (GPPS.SSU). Of them, GGPPS1, GGPPS7, GGPPS8, GPPS.SSU II and GPPS.SSU were translocated mainly into chloroplasts, and GGPPS8 exhibited the optimal catalytic efficiency with respect to catalyzing the formation of GGPP. In addition, the expression pattern of GGPPS8 was similar to that of downstream terpene synthase genes that are directly correlated with triptolide production in roots, indicating that GGPPS8 was most likely to participate in triptolide biosynthesis in roots among the studied enzymes. GPPS.SSU was inactive alone but interacted with GGPPS1, GGPPS7 and GGPPS8 to change the product from GGPP to GPP. These findings implicate that these candidate genes can be regulated to shift the metabolic flux toward diterpenoid formation, increasing the yields of bioactive diterpenoids in plants.

Validation of suitable reference genes for quantitative gene expression analysis in Tripterygium wilfordii.

Validation of suitable reference genes is critical in quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Suitable and reliable reference genes for the normalization of gene expression data are characterized by high gene expression stability across tissues and different experimental conditions. This study evaluated the gene expression stability of ten reference genes commonly used in Arabidopsis thaliana for their suitability in qRT-PCR analysis in Tripterygium wilfordii Hook.f. The orthologous sequences of these ten candidate genes were identified from T. wilfordii transcriptomic data (Project No. SRX472292). Five algorithms including GeNorm, NormFinder, BestKeeper, ΔCt, and RefFinder were used to assess the gene expression stability of these putative reference genes in different plant tissues and different stress conditions. The results identified ACTINT7 and TBP as the most suitable reference genes across all samples. The gene expressions of TwHMGR (3-hydroxy-3-methylglutaryl coenzyme A reductase, KU246037.1) and of TwDXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase, KJ174341.1) were investigated to validate the suitability of the reference genes. The validation analysis confirmed the suitability of ACTINT7 and TBP as the best reference genes for elucidating secondary metabolite biosynthesis pathway in T. wilfordii. In summary, this study identified the most suitable and reliable reference genes for future qRT-PCR- based studies in T. wilfordii.