Feruloyl-CoA 6'-hydroxylase-mediated scopoletin accumulation enhances cotton resistance to Verticillium dahliae.

Verticillium dahliae is a widespread and destructive soilborne fungus that can cause vascular wilt disease and substantially reduce cotton (Gossypium hirsutum) yield and quality. Scopoletin, a natural coumarin, exhibits antifungal activity against V. dahliae; however, the mechanisms of action remain unclear. In this study, we reveal the regulatory activities of feruloyl-CoA 6'-hydroxylase 1 (GhF6'H1) in enhancing V. dahliae resistance by modulating scopoletin accumulation. Silencing GhF6'H1, encoding the pivotal enzyme in scopoletin biosynthesis, through virus-induced silencing resulted in increased susceptibility to V. dahliae and decreased scopoletin accumulation. In transgenic cotton plants expressing GhF6'H1 under the CaMV 35S promoter, GhF6'H1 modulated scopoletin accumulation, affecting cotton resistance to V. dahliae, with increased resistance associated with increased scopoletin accumulation. GhF6'H1 has been identified as a direct target of the transcription factor GhWRKY33-like, indicating that GhWRKY33-like can bind to and activate the GhF6'H1 promoter. Moreover, GhWRKY33-like overexpression in cotton enhanced resistance to V. dahliae through scopoletin accumulation, phenylpropanoid pathway activation, and upregulation of defense response genes. Ectopic expression of GhF6'H1 resulted in effective catalysis of scopoletin synthesis in enzyme assays using substrates like feruloyl coenzyme A, while molecular docking analysis revealed specific amino acid residues playing crucial roles in establishing salt-bridge interactions with the substrate. These findings suggest that GhF6`H1, regulated by GhWRKY33-like, plays a crucial role in enhancing cotton resistance to V. dahliae by modulating scopoletin accumulation.

Transcriptome responses of Arabidopsis to necrotrophic fungus Alternaria brassicae reveal pathways and candidate genes associated with resistance.

Alternaria leaf blight (ALB), caused by a necrotrophic fungus Alternaria brassicae is a serious disease of oleiferous Brassicas resulting in significant yield losses worldwide. No robust resistance against A. brassicae has been identified in the Brassicas. Natural accessions of Arabidopsis show a spectrum of responses to A. brassicae ranging from high susceptibility to complete resistance. To understand the molecular mechanisms of resistance/ susceptibility, we analysed the comparative changes in the transcriptome profile of Arabidopsis accessions with contrasting responses- at different time points post-infection. Differential gene expression, GO enrichment, pathway enrichment, and weighted gene co-expression network analysis (WGCNA) revealed reprogramming of phenylpropanoid biosynthetic pathway involving lignin, hydroxycinnamic acids, scopoletin, anthocyanin genes to be highly associated with resistance against A. brassicae. T-DNA insertion mutants deficient in the biosynthesis of coumarin scopoletin exhibited enhanced susceptibility to A. brassicae. The supplementation of scopoletin to medium or exogenous application resulted in a significant reduction in the A. brassicae growth. Our study provides new insights into the transcriptome dynamics in A. brassicae-challenged Arabidopsis and demonstrates the involvement of coumarins in plant immunity against the Brassica pathogen A. brassicae.

Synergistic induction of phytoalexins in Nicotiana attenuata by jasmonate and ethylene signaling mediated by NaWRKY70.

Production of the phytoalexins scopoletin and scopolin is regulated by jasmonate (JA) and ethylene signaling in Nicotiana species in response to Alternaria alternata, the necrotrophic fungal pathogen that causes brown spot disease. However, how these two signaling pathways are coordinated to control this process remains unclear. In this study, we found that the levels of these two phytoalexins and transcripts of their key enzyme gene, feruloyl-CoA 6'-hydroxylase 1 (NaF6'H1), were synergistically induced in Nicotiana attenuata by co-treatment with methyl jasmonate (MeJA) and ethephon. By combination of RNA sequencing and virus-induced gene silencing, we identified a WRKY transcription factor, NaWRKY70, which had a similar expression pattern to NaF6'H1 and was responsible for A. alternata-induced NaF6'H1 expression. Further evidence from stable transformed plants with RNA interference, knock out and overexpression of NaWRKY70 demonstrated that it is a key player in the synergistic induction of phytoalexins and plant resistance to A. alternata. Electrophoretic mobility shift, chromatin immunoprecipitation-quantitative PCR, and dual-luciferase assays revealed that NaWRKY70 can bind directly to the NaF6'H1 promoter and activate its expression. Furthermore, the key regulator of the ethylene pathway, NaEIN3-like1, can directly bind to the NaWRKY70 promoter and activate its expression. Meanwhile, NaMYC2s, important JA pathway transcription factors, also indirectly regulate the expression of NaWRKY70 and NaF6'H1 to control scopoletin and scopolin production. Our data reveal that these phytoalexins are synergistically induced by JA and ethylene signaling during A. alternata infection, which is largely mediated by NaWRKY70, thus providing new insights into the defense responses against A. alternata in Nicotiana species.

JA/ethylene and NaWRKY6/3 regulated Alternaria resistance depends on ethylene response factor 1B-like in wild tobacco.

Biosynthesis of the phytoalexins scopoletin and scopolin in Nicotiana species is regulated by upstream signals including jasmonate (JA), ethylene (ET) and NaWRKY3 in response to the necrotrophic fungus Alternaria alternata, which causes brown spot disease. However, how these signals are coordinated to regulate these phytoalexins remains unknown. By analyzing RNA sequencing data and RNA interference, we identified NaERF1B-like (NaERF1B-L) as a key player in Nicotiana attenuata during A. alternata infection by regulating the transcripts of Feruloyl-CoA 6'-hydroxylase 1 (NaF6'H1), encoding a key enzyme for scopoletin biosynthesis, and NaVS1-like (NaVS1-L), a putative biosynthetic gene of the phytoalexin solavetivone. We further demonstrated that the synergistic induction of these two genes by JA and ET signaling is mediated by NaERF1B-L. Additionally, we found that the two closely related proteins NaWRKY6 and NaWRKY3 physically interact to enhance NaERF1B-L expression by directly binding and activating the NaERF1B-L promoter. Collectively, our current results demonstrate that NaERF1B-L plays a positive role in resistance to A. alternata by modulating phytoalexins biosynthesis through the integration of JA/ET and NaWRKY6/3 signaling. Our findings reveal a fine-tuned transcriptional regulatory hierarchy mediated by NaERF1B-L for brown spot disease resistance in wild tobacco.

Scopoletin alleviates high glucose-induced toxicity in human renal proximal tubular cells via inhibition of oxidative damage, epithelial-mesenchymal transition, and fibrogenesis.

BACKGROUND: Recent years of evidence suggest the crucial role of renal tubular cells in developing diabetic kidney disease. Scopoletin (SCOP) is a plant-based coumarin with numerous biological activities. This study aimed to determine the effect of SCOP on renal tubular cells in developing diabetic kidney disease and to elucidate mechanisms. METHODS AND RESULTS: In this study, SCOP was evaluated in vitro using renal proximal tubular (HK-2) cells under hyperglycemic conditions to understand its mechanism of action. In HK-2 cells, SCOP alleviated the high glucose-generated reactive oxygen species (ROS), restored the levels of reduced glutathione, and decreased lipid peroxidation. High glucose-induced alteration in the mitochondrial membrane potential was markedly restored in the SCOP-treated cells. Moreover, SCOP significantly reduced the high glucose-induced apoptotic cell population in the Annexin V-FITC flow cytometry study. Furthermore, high glucose markedly elevated the mRNA expression of fibrotic and extracellular matrix (ECM) components, namely, transforming growth factor (TGF)-ß, alfa-smooth muscle actin (α-SMA), collagen I, and collagen III, in HK-2 cells compared to the untreated cells. SCOP treatment reduced these mRNA expressions compared to the high glucose-treated cells. Collagen I and TGF-ß protein levels were also significantly reduced in the SCOP-treated cells. Further findings in HK-2 cells revealed that SCOP interfered with the epithelial-mesenchymal transition (EMT) in the high glucose-treated HK-2 cells by normalizing E-cadherin and downregulating the vimentin and α-SMA proteins. CONCLUSIONS: In conclusion, SCOP modulates the high glucose-generated renal tubular cell oxidative damage and accumulation of ECM components and may be a promising molecule against diabetic nephropathy.

A simple and sensitive high-performance liquid chromatographic method combined with fluorescence detection for bioanalysis of scopoletin in rat plasma: Application to a pharmacokinetic study.

Scopoletin, a coumarin class natural phytoalexin, is present in medicinal plants such as noni (Morinda citrifolia). It exhibits diverse pharmacological properties, including antioxidant, anti-hyperuricemic, and anti-inflammatory effects. The objective of this study was to develop a novel HPLC-fluorescence (HPLC-FL) method for the quantitative analysis of scopoletin in the plasma and to investigate its pharmacokinetics in rats. Sample preparation involved a methanol-based protein precipitation method, and chromatographic separation was conducted using a C18 column with an isocratic mobile phase composed of water and acetonitrile containing 0.1% trifluoroacetic acid. The eluent was detected using an FL detector set to optimized excitation/emission wavelengths of 337/453 nm. Method validation encompassed assessments of selectivity, linearity (1-500 ng/mL), precision, accuracy, recovery, matrix effect, and stability in accordance with the prevailing Food and Drug Administration (FDA) guidelines. The developed method was successfully applied for pharmacokinetic study in rats. To the best of our knowledge, this study is the first application of a simple and sensitive HPLC-FL method for the quantification of scopoletin in a pharmacokinetic study. This method offers a promising alternative for preclinical pharmacokinetic investigations with appropriate modifications and validations and holds potential for clinical applications.

Identification of a Novel Coumarins Biosynthetic Pathway in the Endophytic Fungus Fusarium oxysporum GU-7 with Antioxidant Activity.

Coumarins are generally considered to be produced by natural plants. Fungi have been reported to produce coumarins, but their biosynthetic pathways are still unknown. In this study, Fusarium oxysporum GU-7 and GU-60 were isolated from Glycyrrhiza uralensis, and their antioxidant activities were determined to be significantly different. Abundant dipeptide, phenolic acids, and the plant-derived coumarins fraxetin and scopoletin were identified in GU-7 by untargeted metabolomics, and these compounds may account for its stronger antioxidant activity compared to GU-60. Combined with metabolome and RNA sequencing analysis, we identified 24 potentially key genes involved in coumarin biosynthesis and 6 intermediate metabolites. Interestingly, the best hit of S8H, a key gene involved in hydroxylation at the C-8 position of scopoletin to yield fraxetin, belongs to a plant species. Additionally, nondestructive infection of G. uralensis seeds with GU-7 significantly improved the antioxidant activity of seedlings compared to the control group. This antioxidant activity may depend on the biological characteristics of endophytes themselves, as we observed a positive correlation between the antioxidant activity of endophytic fungi and that of their nondestructively infected seedlings. IMPORTANCE Plant-produced coumarins have been shown to play an important role in assembly of the plant microbiomes and iron acquisition. Coumarins can also be produced by some microorganisms. However, studies on coumarin biosynthesis in microorganisms are still lacking. We report for the first time that fraxetin and scopoletin were simultaneously produced by F. oxysporum GU-7 with strong free radical scavenging abilities. Subsequently, we identified intermediate metabolites and key genes in the biosynthesis of these two coumarins. This is the first report on the coumarin biosynthesis pathway in nonplant species, providing new strategies and perspectives for coumarin production and expanding research on new ways for plants to obtain iron.

NaWRKY3 is a master transcriptional regulator of the defense network against brown spot disease in wild tobacco.

WRKY transcription factors are involved in plant defense against pathogens. No WRKYs have been reported to be involved in resistance to tobacco brown spot disease caused by Alternaria alternata. Here, we found that NaWRKY3 plays a critical role in Nicotiana attenuata defense against A. alternata. NaWRKY3 bound and regulated many defense genes, including: lipoxygenase 3, ACC synthase 1, and ACC oxidase 1, three jasmonate- and ethylene-biosynthetic genes; feruloyl-CoA 6'-hydroxylase 1 (NaF6'H1), the biosynthetic gene for the phytoalexins scopoletin and scopolin; and three A. alternata resistance genes, the long non-coding RNA L2, NADPH oxidase (NaRboh D), and berberine bridge-like (NaBBL28). Silencing L2 reduced jasmonate concentrations and NaF6'H1 expression. NaRboh D-silenced plants were severely impaired in reactive oxygen species production and stomatal closure responses. NaBBL28 was the first A. alternata resistance BBL identified and was involved in the hydroxylation of 17-hydroxygeranyllinalool diterpene glycosides. NaWRKY3 bound to its own promoter but repressed its expression. Thus, we demonstrated that NaWRKY3 is a fine-tuned master regulator of the defense network against A. alternata in N. attenuata by regulating several signaling pathways and defense metabolites. This is the first time such an important WRKY has been identified in Nicotiana species, providing new insights into defense against A. alternata.

Estimation of scopoletin from a polyherbal formulation composition using HPLC coupled with fluorescence detector through the concept of Design of Experiment.

High-performance liquid chromatography (HPLC) coupled with a fluorescence detector was used to analyse bioactive phytoconstituent scopoletin from a polyherbal composition derived from the extract prepared from roots of Argyreia nervosa, roots of Withania somnifera, and fruits of Tribulus terrestris. This analytical method was developed as a quality control tool for standardization of the composition to be formulated to enhance spermatogenesis. Chromatographic separation was achieved using Luna® (250 mm × 4.6 mm, 100 Å, 5 µm) C18 column as a stationary phase, and water (0.01 M glacial acetic acid):methanol: acetonitrile (60:20:20, %v/v/v) as the mobile phase; passed through the column at a set flow rate of 1.0 ml min-1 . The elute in the flow cell was excited at 345 nm and the chromatogram was recorded at 444 nm as the emission wavelength. As a part of the analytical Quality by Design approach, systemic studies were conducted to identify potential risks affecting the critical attributes (area, resolution, retention time) of the analytical method, and mitigating the potential risks after optimizing the chromatographic parameters with the help of the Design of Experiment approach. The developed analytical method was subjected to the validation studies, which showed a linear relationship (r2 = 0.9982) between the concentration and the area corresponding to scopoletin peak in the concentration range 10-130 ng ml-1 . The method was found selective, sensitive, and precise. The recovery of the scopoletin was found in a range 99.53-102.13%; confirming the accuracy of the analytical method. The amount of scopoletin was estimated to be 0.146%w/w from the polyherbal composition.

Scopoletin Reactivates Latent HIV-1 by Inducing NF-κB Expression without Global T Cell Activation.

Reversing HIV-1 latency promotes the killing of infected cells and is essential for cure strategies. However, current latency-reversing agents (LRAs) are not entirely effective and safe in activating latent viruses in patients. In this study, we investigated whether Scopoletin (6-Methoxy-7-hydroxycoumarin), an important coumarin phytoalexin found in plants with multiple pharmacological activities, can reactivate HIV-1 latency and elucidated its underlying mechanism. Using the Jurkat T cell model of HIV-1 latency, we found that Scopoletin can reactivate latent HIV-1 replication with a similar potency to Prostratin and did so in a dose- and time-dependent manner. Moreover, we provide evidence indicating that Scopoletin-induced HIV-1 reactivation involves the nuclear factor kappa B (NF-κB) signaling pathway. Importantly, Scopoletin did not have a stimulatory effect on T lymphocyte receptors or HIV-1 receptors. In conclusion, our study suggests that Scopoletin has the potential to reactivate latent HIV-1 without causing global T-cell activation, making it a promising treatment option for anti-HIV-1 latency strategies.

Design and Synthesis of Scopoletin Sulfonate Derivatives as Potential Insecticidal Agents.

(1) Background: Scopoletin and scoparone, two naturally occurring coumarins, have garnered considerable attention and have been introduced to the market in China due to their high insecticidal efficacy and low toxicity. To investigate the structure-activity relationship of these coumarins, a series of scopoletin derivatives with aryl sulfate at C7 and different substitutes at C3 were designed and synthesized, and their insecticidal activity was studied. (2) Methods: A total of 28 new scopoletin derivatives were designed and synthesized. Most target compounds exhibited moderate insecticidal activity against the phytophagous mite Tetranychus cinnabarinus and the brine shrimp Artemia salina. (3) Results: Among these compounds, compounds 5a and 5j possessed the best insecticidal activities against T. cinnabarinus, with LC50 values of 57.0 and 20.0 µg/mL, respectively, whereas that of the control drug was 15.0 µg/mL. Compound 4j exhibited selective insecticidal activities against A. salina, with an LC50 value of 9.36 µg/mL, whereas its LC50 value against T. cinnabarinus was 93.0 µg/mL. The enzymatic inhibitory activity on acetylcholinesterase (AChE) showed a consistent tendency with the insecticidal activity. Further molecular docking analyses predicted the binding conformations of these compounds, which showed a good correlation between the insecticidal activity and the binding scores. (4) Conclusions: In general, a decreased electron cloud density of the Δ3,4 olefinic bond is beneficial for improving the insecticidal activity against both T. cinnabarinus and A. salina. In addition, naphthyl or benzene groups with a sulfate ester at the C7 position could further improve the insecticidal activity against A. salina. AChE was implied to be a site of action for potential insecticidal activity. The results provide insight into the rational design of a new generation of effective coumarin insecticides.

Scopoletin and umbelliferone protect hepatocytes against palmitate- and bile acid-induced cell death by reducing endoplasmic reticulum stress and oxidative stress.

BACKGROUND: The number of patients with non-alcoholic fatty liver disease (NAFLD) is rapidly increasing due to the growing epidemic of obesity. Non-alcoholic steatohepatitis (NASH), the inflammatory stage of NAFLD, is characterized by lipid accumulation in hepatocytes, chronic inflammation and hepatocyte cell death. Scopoletin and umbelliferone are coumarin-like molecules and have antioxidant, anti-cancer and anti-inflammatory effects. Cytoprotective effects of these compounds have not been described in hepatocytes and the mechanisms of the beneficial effects of scopoletin and umbelliferone are unknown. AIM: To investigate whether scopoletin and/or umbelliferone protect hepatocytes against palmitate-induced cell death. For comparison, we also tested the cytoprotective effect of scopoletin and umbelliferone against bile acid-induced cell death. METHODS: Primary rat hepatocytes were exposed to palmitate (1 mmol/L) or the hydrophobic bile acid glycochenodeoxycholic acid (GCDCA; 50 µmol/L). Apoptosis was assessed by caspase-3 activity assay, necrosis by Sytox green assay, mRNA levels by qPCR, protein levels by Western blot and production of reactive oxygen species (ROS) by fluorescence assay. RESULTS: Both scopoletin and umbelliferone protected against palmitate and GCDCA-induced cell death. Both palmitate and GCDCA induced the expression of ER stress markers. Scopoletin and umbelliferone decreased palmitate- and GCDCA-induced expression of ER stress markers, phosphorylation of the cell death signaling intermediate JNK as well as ROS production. CONCLUSION: Scopoletin and umbelliferone protect against palmitate and bile acid-induced cell death of hepatocytes by inhibition of ER stress and ROS generation and decreasing phosphorylation of JNK. Scopoletin and umbelliferone may hold promise as a therapeutic modality for the treatment of NAFLD.

Cysteine cathepsin C: a novel potential biomarker for the diagnosis and prognosis of glioma.

BACKGROUND: Cysteine cathepsin C encoded by the CTSC gene is an important member of the cysteine cathepsin family that plays a key role regulation of many types of tumors. However, whether CTSC is involved in the pathological process of glioma has not yet been reported. We comprehensively analyzed data from multiple databases and for the first time revealed a role and specific mechanism of action of CTSC in glioma, identifying it as a novel and efficient biomarker for the diagnosis and treatment of this brain tumor. METHODS: The expression of CTSC in glioma and its relationship with clinical characteristics and prognosis of patients with glioma were analyzed at different levels by using clinical sample information from several databases. CTSC expression levels in glioma and normal brain tissues, as well as in glioma cells and normal brain cells, was validated by real-time quantitative polymerase chain reaction (RT-qPCR). Gene set enrichment analysis (GSEA) was used to reveal the signaling pathways that CTSC may participate in. The connectivity map was used to reveal small molecules that may inhibit CTSC expression in glioma, and the putative effect of these compounds was verified by RT-qPCR. RESULTS: Our analyses showed that the expression of CTSC in glioma was higher than that in non-cancerous cells. GSEA showed that CTSC expression may regulate the malignant development of glioma through Toll-like receptor signaling pathways, pathways in cancer, and extracellular matrix receptor interaction signaling pathways. And we proved piperlongumine and scopoletin could inhibit CTSC expression in glioma cells. CONCLUSIONS: CTSC may serve as an efficient molecular target for the diagnosis and therapy of glioma, thereby improving the poor prognosis of patients with glioma.

Shortened lifespan induced by a high-glucose diet is associated with intestinal immune dysfunction in Drosophila sechellia.

Organisms can generally be divided into two nutritional groups: generalists that consume various types of food and specialists that consume specific types of food. However, it remains unclear how specialists adapt to only limited nutritional conditions in nature. In this study, we addressed this question by focusing on Drosophila fruit flies. The generalist Drosophila melanogaster can consume a wide variety of foods that contain high glucose levels. In contrast, the specialist Drosophila sechellia consumes only the Indian mulberry, known as noni (Morinda citrifolia), which contains relatively little glucose. We showed that the lifespan of D. sechellia was significantly shortened under a high-glucose diet, but this effect was not observed for D. melanogaster. In D. sechellia, a high-glucose diet induced disorganization of the gut epithelia and visceral muscles, which was associated with abnormal digestion and constipation. RNA-sequencing analysis revealed that many immune-responsive genes were suppressed in the gut of D. sechellia fed a high-glucose diet compared with those fed a control diet. Consistent with this difference in the expression of immune-responsive genes, high glucose-induced phenotypes were restored by the addition of tetracycline or scopoletin, a major nutritional component of noni, each of which suppresses gut bacterial growth. We propose that, in D. sechellia, a high-glucose diet impairs gut immune function, which leads to a change in gut microbiota, disorganization of the gut epithelial structure and a shortened lifespan.

An overview of the pharmacological activities of scopoletin against different chronic diseases.

Chronic diseases are considered a major public health concern worldwide, and most of these diseases like cancer, cardiovascular, metabolic, and neurological disorders occur due to atypical regulation of multiple signaling pathways. It has also been observed that most of the currently approved therapies for these diseases fail to show prolonged efficacy due to their mono-targeted nature and are associated with the development of chemoresistance, thus restricting their utility. The plant-derived compounds, on the other hand, show multi-targeted nature, and thus these phytochemicals have gained wide attention as they offer negligible side effects. The present review aims to recapitulate the potential effects of one such phytochemical, Scopoletin, which was found to have a diverse range of pharmacological activities such as anti-cancer, anti-diabetic, anti-inflammatory, cardioprotective, hepatoprotective, etc. Scopoletin modulated multiple molecular signatures in cancer, including AMPK, EGFR, MAPK/ ERK, NF-κB, PI3K/Akt/ mTOR, and STAT3; regulated the levels of critical markers of metabolic diseases such as ALT, AST, TG, and TC; inflammatory diseases such as ILs and TNFs; neurological diseases such as AChE, etc. thus relieving the symptoms and severity associated with these diseases. Further, this compound has a non-toxic nature and possesses an excellent pharmacokinetic property, which warrants further investigation in clinical settings for developing it as a potential drug.

Advances in biosynthesis of scopoletin.

Scopoletin is a typical example of coumarins, which can be produced in plants. Scopoletin acts as a precursor for pharmaceutical and health care products, and also possesses promising biological properties, including antibacterial, anti-tubercular, anti-hypertensive, anti-inflammatory, anti-diabetic, and anti-hyperuricemic activity. Despite the potential benefits, the production of scopoletin using traditional extraction processes from plants is unsatisfactory. In recent years, synthetic biology has developed rapidly and enabled the effective construction of microbial cell factories for production of high value-added chemicals. Herein, this review summarizes the progress of scopoletin biosynthesis in artificial microbial cell factories. The two main pathways of scopoletin biosynthesis are summarized firstly. Then, synthetic microbial cell factories are reviewed as an attractive improvement strategy for biosynthesis. Emerging techniques in synthetic biology and metabolic engineering are introduced as innovative tools for the efficient synthesis of scopoletin. This review showcases the potential of biosynthesis of scopoletin in artificial microbial cell factories.

Knockdown of p-Coumaroyl Shikimate/Quinate 3'-Hydroxylase Delays the Occurrence of Post-Harvest Physiological Deterioration in Cassava Storage Roots.

Cassava storage roots are an important source of food, feed, and material for starch-based industries in many countries. After harvest, rapid post-harvest physiological deterioration (PPD) reduces their palatability and marketability. During the PPD process, vascular streaking occurs through over-accumulation of coumarins, the biosynthesis of which involves the key enzyme p-coumaroyl shikimate/quinate 3'-hydroxylase (C3'H). Repression of MeC3'H expression by RNA interference in transgenic cassava plants caused a significant delay in PPD by decreasing scopoletin and scopolin accumulation in field-harvested storage roots. This study demonstrates that MeC3'H is the key enzyme participating in coumarin biosynthesis during PPD and shows that MeC3'H is a useful target gene for editing to prolong the shelf life of cassava storage roots.

Challenges in the Heterologous Production of Furanocoumarins in Escherichia coli.

Coumarins and furanocoumarins are plant secondary metabolites with known biological activities. As they are present in low amounts in plants, their heterologous production emerged as a more sustainable and efficient approach to plant extraction. Although coumarins biosynthesis has been positively established, furanocoumarin biosynthesis has been far more challenging. This study aims to evaluate if Escherichia coli could be a suitable host for furanocoumarin biosynthesis. The biosynthetic pathway for coumarins biosynthesis in E. coli was effectively constructed, leading to the production of umbelliferone, esculetin and scopoletin (128.7, 17.6, and 15.7 µM, respectively, from tyrosine). However, it was not possible to complete the pathway with the enzymes that ultimately lead to furanocoumarins production. Prenyltransferase, psoralen synthase, and marmesin synthase did not show any activity when expressed in E. coli. Several strategies were tested to improve the enzymes solubility and activity with no success, including removing potential N-terminal transit peptides and expression of cytochrome P450 reductases, chaperones and/or enzymes to increase dimethylallylpyrophosphate availability. Considering the results herein obtained, E. coli does not seem to be an appropriate host to express these enzymes. However, new alternative microbial enzymes may be a suitable option for reconstituting the furanocoumarins pathway in E. coli. Nevertheless, until further microbial enzymes are identified, Saccharomyces cerevisiae may be considered a preferred host as it has already been proven to successfully express some of these plant enzymes.

Scopoletin protects retinal ganglion cells 5 from high glucose-induced injury in a cellular model of diabetic retinopathy via ROS-dependent p38 and JNK signaling cascade.

The protective activity of scopoletin (SPT) against glucose-induced cataract has been attributed to attenuation of aldose reductase activity and oxidative stress in a rat model. The present investigation was aimed to study the protective effect and mechanism of SPT in retinal ganglia cells (RGC) under oxidative stress and apoptosis induced by hyperglycemia. The RGC-5 cells were pre-conditioned with variable SPT concentrations for 6 hours and then subjected to hyperglycemia for 48 hours. The cell viability, mito- chondrial membrane potential (MMP) and oxidative stress markers were quantified. Western blotting was employed to screen the expression of mitogen-activated protein kinase (MAPK) and various apoptosis related proteins. SPT blocked the high-glucose induced cell injury and normalized the mitochondrial functioning via lowering the loss of MMP and release of cytochrome c. Pretreatment with SPT suppressed the enhanced ROS, malondialdehyde, and protein carbonyl content triggered by high-glucose exposure in RGC-5 cells. SPT normalized the apoptotic proteins in RGC-5 cells. The phosphorylation of c-Jun N-terminal kinases (JNK) and p38 MAPK in RGC-5 due to hyperglycemia was attenuated by SPT. Overall, SPT exhibited a protective effect in RGC-5 cells exposed to a high-glucose environment via its antioxidant efficacy, inhibition of apoptosis and modulation of the ROS-dependent p38/JNK signaling cascade.

Scopoletin Attenuates Intracerebral Hemorrhage-Induced Brain Injury and Improves Neurological Performance in Rats.

BACKGROUND: Among the hypertension-related complications, the onset of intracerebral hemorrhage (ICH) is a destructive stage and is the most disabling type of stroke that has the highest death rate. At present, there is no promising treatment for ICH. OBJECTIVES: The present investigation was aimed at evaluating the safeguarding effect of scopoletin against ICH-induced brain injury. METHODS: We used Wistar male rats and divided them into 4 groups. Group 1 served as control, group 2 was induced with ICH, group 3 served as scopoletin-pretreated ICH rats, and group 4 as scopoletin drug control. During the experimental period, neurobehavioral outcome, cerebral edema, and neuroinflammation parameters were evaluated using RT-PCR and other biochemical analyses. RESULTS: The rats that received scopoletin treatment demonstrated a significant attenuation in neurological deficits, neurodegeneration markers expression (TREM-1, SERPINE-1), and restored cerebral edema compared to ICH animals. On the other hand, an upsurge in inflammatory cytokines, for example, TNF-α, IL-13, IL-1ß, and IL-17, was observed in ICH rats and was reduced to the level near normalcy in the scopoletin-treated groups. CONCLUSION: Our investigations propose that the effectiveness of scopoletin in improving acute neurological function after ICH is promising, and this could be a lead molecule for the development of treatment plans in ICH treatment.