IMP: bridging the gap for medicinal plant genomics.

Medicinal plants have garnered significant attention in ethnomedicine and traditional medicine due to their potential antitumor, anti-inflammatory and antioxidant properties. Recent advancements in genome sequencing and synthetic biology have revitalized interest in natural products. Despite the availability of sequenced genomes and transcriptomes of these plants, the absence of publicly accessible gene annotations and tabular formatted gene expression data has hindered their effective utilization. To address this pressing issue, we have developed IMP (Integrated Medicinal Plantomics), a freely accessible platform at https://www.bic.ac.cn/IMP. IMP curated a total of 8 565 672 genes for 84 high-quality genome assemblies, and 2156 transcriptome sequencing samples encompassing various organs, tissues, developmental stages and stimulations. With the integrated 10 analysis modules, users could simply examine gene annotations, sequences, functions, distributions and expressions in IMP in a one-stop mode. We firmly believe that IMP will play a vital role in enhancing the understanding of molecular metabolic pathways in medicinal plants or plants with medicinal benefits, thereby driving advancements in synthetic biology, and facilitating the exploration of natural sources for valuable chemical constituents like drug discovery and drug production.

Identification and characterization of two Isatis indigotica O-methyltransferases methylating C-glycosylflavonoids.

Isatis indigotica accumulates several active substances, including C-glycosylflavonoids, which have important pharmacological activities and health benefits. However, enzymes catalyzing the methylation step of C-glycosylflavonoids in I. indigotica remain unknown. In this study, three O-methyltransferases (OMTs) were identified from I. indigotica that have the capacity for O-methylation of the C-glycosylflavonoid isoorientin. The Type II OMTs IiOMT1 and IiOMT2 efficiently catalyze isoorientin to form isoscoparin, and decorate one of the aromatic vicinal hydroxyl groups on flavones and methylate the C6, C8, and 3'-hydroxyl positions to form oroxylin A, wogonin, and chrysoeriol, respectively. However, the Type I OMT IiOMT3 exhibited broader substrate promiscuity and methylated the C7 and 3'-hydroxyl positions of flavonoids. Further site-directed mutagenesis studies demonstrated that five amino acids of IiOMT1/IiOMT2 (D121/D100, D173/D149, A174/A150R, N200/N176, and D248/D233) were critical residues for their catalytic activity. Additionally, only transient overexpression of Type II OMTs IiOMT1 and IiOMT2 in Nicotiana benthamiana significantly increased isoscoparin accumulation, indicating that the Type II OMTs IiOMT1 and IiOMT2 could catalyze the methylation step of C-glycosylflavonoid, isoorientin at the 3'-hydroxyl position. This study provides insights into the biosynthesis of methylated C-glycosylflavonoids, and IiOMTs could be promising catalysts in the synthesis of bioactive compounds.

Network Pharmacology Research and Dual-omic Analyses Reveal the Molecular Mechanism of Natural Product Nodosin Inhibiting Muscle-Invasive Bladder Cancer in Vitro and in Vivo.

Bladder cancer, specifically, muscle-invasive bladder cancer (MIBC), is among the most common malignant tumors. Patients with MIBC who cannot tolerate standard drugs require novel treatments. Targeting apoptosis may help treat cancer, which may be achieved with the use of some natural products. Nodosin, found in Isodon serra (Maxim.) Kudo (known as Xihuangcao), may inhibit bladder cancer cells. Transcriptomics and proteomics dual-omic analyses revealed the network pharmacological mechanism: (1) blocking the S phase by up-regulating RPA2, CLSPN, MDC1, PDCD2L, and E2F6 gene expressions, suppressing cancer cell proliferation; (2) inducing apoptosis and autophagy and restraining ferroptosis by up-regulating HMOX1, G0S2, SQSTM1, FTL, SLC7A11, and AIFM2 gene expressions; (3) preventing cancer cell migration by down-regulating NEXN, LIMA1, CFL2, PALLD, and ITGA3 gene expressions. In vivo, nodosin inhibited bladder cancer cell growth in a model of xenograft tumor in nude mice. This study is the first to report basic research findings on the network pharmacological mechanism of cytotoxicity of bladder cancer cells by nodosin, providing novel evidence for the application of nodosin in the field of oncology; however, other mechanisms may be involved in the effects of nodosin for further research. These findings provide a foundation for the development of novel MIBC drugs.

TMT-Based Quantitative Proteomics Analysis Reveals the Panoramic Pharmacological Molecular Mechanism of ß-Elemonic Acid Inhibition of Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common cancers worldwide but has limited available therapeutic methods; therefore, there is a need to develop highly efficient prevention and treatment strategies. Here, we investigated the anti-cancer activity of ß-elemonic acid (EA) in CRC in vitro and in vivo. Our results showed that EA inhibited cell proliferation and migration in the CRC cell lines SW480 and HCT116. Moreover, EA significantly suppressed the growth of transplanted colorectal tumors in nude mice. Interestingly, high-throughput tandem mass tag (TMT)-based quantitative proteomics indicated that EA mainly targets tumor mitochondria and attenuates the translation of 54 mitochondrial ribosome proteins, many of which are discovered significantly upregulated in clinical CRC patients. More interestingly, EA at a low concentration (lower than 15 µg/ml) repressed the cell cycle by downregulating CDK1, CDK6, and CDC20, whereas at a high concentration (higher than 15 µg/ml), caused a non-apoptotic cell death-ferroptosis via downregulating ferritin (FTL) and upregulating transferrin (TF), ferroxidase (CP), and acyl-CoA synthetase long-chain family member 4 (ACSL4). This is the first report on the panoramic molecular mechanism of EA against CRC, which would make great contributions to developing a novel drug for colorectal cancer therapy.

Elucidation of the essential oil biosynthetic pathways in Cinnamomum burmannii through identification of six terpene synthases.

Cinnamomum burmannii is a traditional plant that has long been used as a spice, food preservative, and food flavoring. Essential oils in C. burmannii, which mainly consist of mono- and sesquiterpenes such borneol, linalool, and caryophyllene, have impressive pharmaceutical properties. Although the transcriptome-based discovery of (+)-bornyl diphosphate synthase (CbTPS1) from C. burmannii was reported in our previous study, the remaining terpene synthases (TPSs) corresponding to various terpene biosynthesis pathways remain unidentified. In this study, we report the results of RNA-sequencing of a borneol type plant and functional characterization of six additional full-length candidate TPS genes (named CbTPS2-7). Phylogenetic analysis revealed that CbTPS2 and CbTPS3 together with the previously identified CbTPS1 protein belong to the TPS-b subfamily, and enzyme assays using geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) as substrates revealed that CbTPS1, CbTPS2 and CbTPS3 catalyze the formation of monoterpenes. CbTPS4, CbTPS5, and CbTPS6, which belong to the TPS-a clade, generated monoterpenes and sesquiterpenes. CbTPS7, which belongs to the TPS-g clade, showed linalool/nerolidol synthase activity. These CbTPSs identified in C. burmannii produced a total of 10 monoterpenes and 14 sesquiterpenes in an in vitro assay. These findings clarify the biosynthesis pathways of 13 monoterpenoids and 12 sesquiterpenoids in the leaf essential oil of C. burmannii and shed light on terpene biosynthesis in Cinnamomum.

Characterization of O-methyltransferases involved in the biosynthesis of tetrandrine in Stephania tetrandra.

Tetrandrine is the most effective small molecule that has been found to inhibit the Ebola virus. It is a typical bisbenzylisoquinoline alkaloid and is the main active ingredient in Stephania tetrandra. Metabolic engineering and synthetic biology are potential methods for efficient and rapid acquisition of tetrandrine. S-adenosyl-L-methionine: (S)-norcoclaurine-6-O-methyltransferase (6OMT) is a rate-limiting step involved in the biosynthesis of tetrandrine. In this study, we identify S-adenosyl-L-methionine: (S)-norcoclaurine-6-O-methyltransferase from S. tetrandra, which catalyzes the conversion of (S)-norcoclaurine to (S)-coclaurine. Four 6OMT-like genes were cloned from S. tetrandra. An in vitro enzyme assay showed that St6OMT1 could catalyze the conversion of (S)-norcoclaurine to produce (S)-coclaurine. St6OMT2 can catalyze the production of very few (S)-coclaurine molecules, accompanied by more by-products with m/z 300, compared to St6OMT1. The newly discovered 6OMTs will provide an optional genetic component for benzylisoquinoline alkaloid (BIA) synthetic biology research. This work will lay the foundation for the analysis of the biosynthetic pathway of tetrandrine in S. tetrandra.

Transcriptomic Insight into Terpenoid Biosynthesis and Functional Characterization of Three Diterpene Synthases in Scutellaria barbata.

Scutellaria barbata (Lamiaceae) is an important medicinal herb widely used in China, Korea, India, and other Asian countries. Neo-clerodane diterpenoids are the largest known group of Scutellaria diterpenoids and show promising cytotoxic activity against several cancer cell lines. Here, Illumina-based deep transcriptome analysis of flowers, the aerial parts (leaf and stem), and roots of S. barbata was used to explore terpenoid-related genes. In total, 121,958,564 clean RNA-sequence reads were assembled into 88,980 transcripts, with an average length of 1370 nt and N50 length of 2144 nt, indicating high assembly quality. We identified nearly all known terpenoid-related genes (33 genes) involved in biosynthesis of the terpenoid backbone and 14 terpene synthase genes which generate skeletons for different terpenoids. Three full length diterpene synthase genes were functionally identified using an in vitro assay. SbTPS8 and SbTPS9 were identified as normal-CPP and ent-CPP synthase, respectively. SbTPS12 reacts with SbTPS8 to produce miltiradiene. Furthermore, SbTPS12 was proven to be a less promiscuous class I diterpene synthase. These results give a comprehensive understanding of the terpenoid biosynthesis in S. barbata and provide useful information for enhancing the production of bioactive neo-clerodane diterpenoids through genetic engineering.

Structural characterization and discrimination of the Paris polyphylla var. yunnanensis and Paris vietnamensis based on metabolite profiling analysis.

This study aimed to distinguish the rhizomes of Paris polyphylla var. yunnanensis (Franch) Hand Mazz (PPY) and Paris veitnamensis (Takht.) H. Li (PV) using metabolomics-based ultra high-performance liquid chromatography coupled with quadrupole time-of-fligh mass spectrometry (UHPLC/Q-TOF MS). First, the UHPLC/Q-TOF MS approach was optimized for metabolite profiling. Then, the MS data were processed using UNIFI™ combined with an in-house library to automatically characterize the metabolites. Based on the exact mass information, the fragmentation characteristics, and the retention time of compounds, and the fragmentation mechanism and retention behavior of steroidal glycosides in the references, the structures identified by UNIFI were further verified. Overall, 146 metabolites, including 42 potential new compounds, were identified or tentatively identified. Pattern recognition analysis of the PPY and PV MS data revealed that they were clearly separated, and 15 potential biomarkers for differentiating between them were selected. These biomarkers were subsequently used to successfully predict the genus of PPY and PV samples. These results indicated that metabolite profiling by UHPLC/Q-TOF MS is an effective, robust approach for determining the characteristic biomarkers that differentiate between TCM species with multiple botanical origins.

Functional Diversification of Kaurene Synthase-Like Genes in Isodon rubescens.

Ent-kaurene diterpenoids are the largest group of known Isodon diterpenoids. Among them, oridonin is accumulated in the leaves, and is the most frequently studied compound because of its antitumor and antibacterial activities. We have identified five copalyl diphosphate synthase (CPS) and six kaurene synthase-like (KSL) genes by transcriptome profiling of Isodon rubescens leaves. An in vitro assay assigns ten of them to five different diterpene biosynthesis pathways, except IrCPS3 that has a mutation in the catalytic motif. The Lamiaceae-specific clade genes (IrCPS1 and IrCPS2) synthesize the intermediate copalyl diphosphate (normal-CPP), while IrCPS4 and IrCPS5 synthesize the intermediate ent-copalyl diphosphate (ent-CPP). IrKSL2, IrKSL4, and IrKSL5 react with ent-CPP to produce an ent-isopimaradiene-like compound, ent-atiserene and ent-kaurene, respectively. Correspondingly, the Lamiaceae-specific clade genes IrKSL1 or IrKSL3 combined with normal-CPP led to the formation of miltiradiene. The compound then underwent aromatization and oxidization with a cytochrome P450 forming two related compounds, abietatriene and ferruginol, which were detected in the root bark. IrKSL6 reacts with normal-CPP to produce isopimaradiene. IrKSL3 and IrKSL6 have the γßα tridomain structure, as these proteins tend to possess the bidomain structure of IrKSL1, highlighting the evolutionary history of KSL gene domain loss and further elucidating chemical diversity evolution from a macroevolutionary stance in Lamiaceae.

Effects of different doses of cadmium on secondary metabolites and gene expression in Artemisia annua L.

This study aims to elucidate the underlying molecular mechanisms of artemisinin accumulation induced by Cd. The effects of different Cd concentrations (0, 20, 60, and 120 µmol/L) on the biosynthesis of Artemisia annua L. were examined. Intermediate and end products were quantified by HPLC-ESI-MS/MS analysis. The expression of key biosynthesis enzymes was also determined by qRT-PCR. The results showed that the application of treatment with 60 and 120 µmol/L Cd for 3 days significantly improved the biosynthesis of artemisinic acid, arteannuin B, and artemisinin. The concentrations of artemisinic acid, arteannuin B, and artemisinin in the 120 µmol/L Cd-treated group were 2.26, 102.08, and 33.63 times higher than those in the control group, respectively. The concentrations of arteannuin B and artemisinin in 60 µmol/L Cd-treated leaves were 61.10 and 26.40 times higher than those in the control group, respectively. The relative expression levels of HMGR, FPS, ADS, CYP71AV1, DBR2, ALDH1, and DXR were up-regulated in the 120 µmol/L Cd-treated group because of increased contents of artemisinic metabolites after 3 days of treatment. Hence, appropriate doses of Cd can increase the concentrations of artemisinic metabolites at a certain time point by up-regulating the relative expression levels of key enzyme genes involved in artemisinin biosynthesis.

[Mitogen-activated protein kinase genes of Artemisia annua and their expression analysis under Cadmium stress].

In order to study Artemisia annua under cadmium stress, whether there are corresponding MAPK genes involved in transduction of the cadmium signal. 17 AaMAPK genes, named AaMAPK1-AaMAPK17 repectively, were finally obtained by using Trinity method for de novo assembly of transcripts from SRA database and BLAST search against AtMAPK genes and determing conserved domain using a series of bioinformatics tools. There exist 16 MAPK genes contained T[D/E]Y conserved domains among the obtained genes. The expressions of these genes were analyzed by Real-time PCR under cadmium stress. The results showed that the expressions level of AaMAPK3 and AaMAPK10 were down-regulated and MAPK7, MAPK9 and MAPK12 were up-regulated. These indicated that there exist corresponding MAPK genes involved in transduction of the cadmium signal.

[Cloning and expression analysis of a hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferasegene(IiHCT) from Isatis indigotica].

Based on the transcriptome data, we cloned the open reading frame of IiHCT gene from Isatis indigotica, and then performed bioinformatic analysis of the sequence. Further, we detected expression pattern in specific organs and hairy roots treated methyl jasmonate( MeJA) by RT-PCR. The IiHCT gene contains a 1 290 bp open reading frame( ORF) encoding a polypeptide of 430 amino acids. The predicted isoelectric point( pI) was 5.7, a calculated molecular weight was about 47.68 kDa. IiHCT was mainly expressed in stem and undetectable in young root, leaf and flower bud. After the treatment of MeJA, the relative expression level of IiHCT increased rapidly. The expression level of IiHCT was the highest at 4 h and maintained two fold to control during 24 h. In this study, cloning of IiHCT laid the foundation for illustrating the biosynthesis mechanism of phenylpropanoids in I. indigotica.

[Stability analysis of reference gene based on real-time PCR in Artemisia annua under cadmium treatment].

In this study, Actin, 18S rRNA, PAL, GAPDH and CPR of Artemisia annua were selected as candidate reference genes, and their gene-specific primers for real-time PCR were designed, then geNorm, NormFinder, BestKeeper, Delta CT and RefFinder were used to evaluate their expression stability in the leaves of A. annua under treatment of different concentrations of Cd, with the purpose of finding a reliable reference gene to ensure the reliability of gene-expression analysis. The results showed that there were some significant differences among the candidate reference genes under different treatments and the order of expression stability of candidate reference gene was Actin > 18S rRNA > PAL > GAPDH > CPR. These results suggested that Actin, 18S rRNA and PAL could be used as ideal reference genes of gene expression analysis in A. annua and multiple internal control genes were adopted for results calibration. In addition, differences in expression stability of candidate reference genes in the leaves of A. annua under the same concentrations of Cd were observed, which suggested that the screening of candidate reference genes was needed even under the same treatment. To our best knowledge, this study for the first time provided the ideal reference genes under Cd treatment in the leaves of A. annua and offered reference for the gene expression analysis of A. annua under other conditions.

HDAC6 deacetylates and ubiquitinates MSH2 to maintain proper levels of MutSα.

MutS protein homolog 2 (MSH2) is a key DNA mismatch repair protein. It forms the MSH2-MSH6 (MutSα) and MSH2-MSH3 (MutSß) heterodimers, which help to ensure genomic integrity. MutSα not only recognizes and repairs mismatched nucleotides but also recognizes DNA adducts induced by DNA-damaging agents, and triggers cell-cycle arrest and apoptosis. Loss or depletion of MutSα from cells leads to microsatellite instability (MSI) and resistance to DNA damage. Although the level of MutSα can be reduced by the ubiquitin-proteasome pathway, the detailed mechanisms of this regulation remain elusive. Here we report that histone deacetylase 6 (HDAC6) sequentially deacetylates and ubiquitinates MSH2, leading to MSH2 degradation. In addition, HDAC6 significantly reduces cellular sensitivity to DNA-damaging agents and decreases cellular DNA mismatch repair activities by downregulation of MSH2. Overall, these findings reveal a mechanism by which proper levels of MutSα are maintained.

A novel function of the Fe65 neuronal adaptor in estrogen receptor action in breast cancer cells.

Fe65 is a multidomain adaptor with established functions in neuronal cells and neurodegeneration diseases. It binds to the C terminus of the Aß amyloid precursor protein and is involved in regulating gene transcription. The present studies show that Fe65 is expressed in breast cancer (BCa) cells and acts as an ERα transcriptional coregulator that is recruited by 17ß-estradiol to the promoters of estrogen target genes. Deletion analyses mapped the ERα binding domain to the phosphotyrosine binding domain 2 (PTB2). Ectopic Fe65 increased the transcriptional activity of the ERα in a PTB2-dependent manner in reporter assays. Fe65 knockdown decreased, whereas its stable expression increased the transcriptional activity of endogenous ERα in BCa cells and the ability of estrogens to stimulate target gene expression, ERα, and coactivator recruitment to target gene promoters and cell growth. Furthermore, Fe65 expression decreased the antagonistic activity of tamoxifen (TAM), suggesting a role for Fe65 in TAM resistance. Overall, the studies define a novel role for the neuronal adaptor in estrogen actions in BCa cells.

1,25-Dihydroxyvitamin D3 suppresses telomerase expression and human cancer growth through microRNA-498.

Telomerase is an essential enzyme that counteracts the telomere attrition accompanying DNA replication during cell division. Regulation of the promoter activity of the gene encoding its catalytic subunit, the telomerase reverse transcriptase, is established as the dominant mechanism conferring the high telomerase activity in proliferating cells, such as embryonic stem and cancer cells. This study reveals a new mechanism of telomerase regulation through non-coding small RNA by showing that microRNA-498 (miR-498) induced by 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) decreases the mRNA expression of the human telomerase reverse transcriptase. MiR-498 was first identified in a microarray analysis as the most induced microRNA by 1,25(OH)(2)D(3) in ovarian cancer cells and subsequently validated by quantitative polymerase chain reaction assays in multiple human cancer types. A functional vitamin D response element was defined in the 5-prime regulatory region of the miR-498 genome, which is occupied by the vitamin D receptor and its coactivators. Further studies showed that miR-498 targeted the 3-prime untranslated region of human telomerase reverse transcriptase mRNA and decreased its expression. The levels of miR-498 expression were decreased in malignant human ovarian tumors as well as human ovarian cancer cell lines. The ability of 1,25(OH)(2)D(3) to decrease human telomerase reverse transcriptase mRNA and to suppress ovarian cancer growth was compromised when miR-498 was depleted using the sponges in cell lines and mouse tumor models. Taken together, our studies define a novel mechanism of telomerase regulation by small non-coding RNAs and identify miR-498 as an important mediator for the anti-tumor activity of 1,25(OH)(2)D(3).

The coupling of epidermal growth factor receptor down regulation by 1alpha,25-dihydroxyvitamin D3 to the hormone-induced cell cycle arrest at the G1-S checkpoint in ovarian cancer cells.

1alpha,25-dihydroxyvitamin D3, 1,25(OH)(2)D(3), regulates gene expression through the vitamin D receptor. The present studies identify the epidermal growth factor receptor, EGFR, as a target gene suppressed by 1,25(OH)(2)D(3) in human ovarian cancer cells. The suppression was detected at both mRNA and protein levels in vitamin D-sensitive human ovarian cancer cells. A novel vitamin D response element was identified in intron 1 of the EGFR genome, a known hotspot for its transcriptional regulation. Chromatin immunoprecipitations and reporter gene analyses showed that the intronic DNA element bound to vitamin D receptor and a co-repressor and was functional in mediating transcriptional suppression of EGFR promoter by 1,25(OH)(2)D(3) under stable transfection conditions. Consistent with the EGFR down regulation, 1,25(OH)(2)D(3) suppressed activation of the external signal regulated kinase by epidermal growth factors. Over expression of an active EGFR in vitamin D sensitive ovarian cancer cells caused resistance to 1,25(OH)(2)D(3)-induced growth suppression and diminished the hormonal regulation of cyclin D1, cyclin E, Skp2 and p27, a group of cell cycle regulators that mediate 1,25(OH)(2)D(3)-induced cell cycle arrest at G1-S checkpoint. Taken together, our studies demonstrate that 1,25(OH)(2)D(3) suppresses the response of human ovarian cancer cells to mitogenic growth factors and couple the suppression to the cell cycle arrest at G1-S checkpoint by the hormone.