[Accumulation and biosynthesis mechanism of volatile oils in glandular scale of Agastache rugosa].

The volatile oils are the effective components of Agastache rugosa, which are stored in the glandular scale. The leaves of pulegone-type A. rugosa were used as materials to observe the leaf morphology of A. rugosa at different growth stages, and the components of volatile oils in gland scales were detected by GC-MS. At the same time, qRT-PCR was used to determine the relative expression of key enzyme genes in the biosynthesis pathway of monoterpenes in volatile oils. The results showed that the density of A. rugosa glandular scale decreased first and then tended to be stable. With the growth of leaves, the relative content of pulegone decreased from 79.26% to 3.94%(89.97%-41.44%), while that of isomenthone increased from 2.43% to 77.87%(0.74%-51.01%), and the changes of other components were relatively insignificant. The correlation analysis between the relative content of monoterpenes and the relative expression levels of their key enzyme genes showed that there was a significant correlation between the relative content of menthone and isomenthone and the relative expression levels of pulegone reductase(PR)(r>0.6, P<0.01). To sum up, this study revealed the accumulation rules of the main components of the contents of the glandular scale of A. rugosa and the expression rules of the key enzyme genes for biosynthesis, which provided a scientific basis and data support for determining the appropriate harvesting period and quality control of the medicinal herbs. This study also initially revealed the biosynthesis mechanism of the monoterpenes mainly composed of pulegone and isomenthone in A. rugosa, laying a foundation for further research on the molecular mechanism of synthesis and accumulation of monoterpenes in A. rugosa.

Adjusting function of camphor on primary metabolism in Cinnamomum camphora stressed by high temperature.

Cinnamomum camphora has great economic value for its wide utilization in traditional medicine and furniture material, and releases lots of monoterpenes to tolerate high temperature. To uncover the adjusting function of monoterpenes on primary metabolism and promoting their utilization as anti-high temperature agents, the photosynthetic capacities, primary metabolite levels, cell ultrastructure and associated gene expression were surveyed in C. camphora when it was blocked monoterpene biosynthesis with fosmidomycin (Fos) and fumigated with camphor (a typical monoterpene in the plant) under high temperature (Fos+38 °C+camphor). Compared with the control (28 °C), high temperature at 38 °C decreased the starch content and starch grain size, and increased the fructose, glucose, sucrose and soluble sugar content. Meanwhile, high temperature also raised the lipid content, with the increase of lipid droplet size and numbers. These variations were further intensified in Fos+ 38 °C treatment. Compared with Fos+ 38 °C treatment, Fos+ 38 °C+camphor treatment improved the starch accumulation by promoting 4 gene expression in starch biosynthesis, and lowered the sugar content by suppressing 3 gene expression in pentose phosphate pathway and promoting 15 gene expression in glycolysis and tricarboxylic acid cycle. Meanwhile, Fos+ 38 °C+camphor treatment also lowered the lipid content, which may be caused by the down-regulation of 2 genes in fatty acid formation and up-regulation of 4 genes in fatty acid decomposition. Although Fos+ 38 °C+camphor treatment improved the photosynthetic capacities in contrast to Fos+ 38 °C treatment, it cannot explain the variations of these primary metabolite levels. Therefore, camphor should adjust related gene expression to maintain the primary metabolism in C. camphora tolerating high temperature.

Combined in†vitro/in silico approaches, molecular dynamics simulations and safety assessment of the multifunctional properties of thymol and carvacrol: A comparative insight.

Bioactive compounds derived from medicinal plants have acquired immense attentiveness in drug discovery and development. The present study investigated in vitro and predicted in silico the antibacterial, antifungal, and antiviral properties of thymol and carvacrol, and assessed their safety. The performed microbiological assays against Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica Typhimurium revealed that the minimal inhibitory concentration values ranged from (0.078 to 0.312 mg/mL) and the minimal fungicidal concentration against Candida albicans was 0.625 mg/mL. Molecular docking simulations, stipulated that these compounds could inhibit bacterial replication and transcription functions by targeting DNA and RNA polymerases receptors with docking scores varying between (-5.1 to -6.9 kcal/mol). Studied hydroxylated monoterpenes could hinder C. albicans growth by impeding lanosterol 14α-demethylase enzyme and showed a (ΔG=-6.2 and -6.3 kcal/mol). Computational studies revealed that thymol and carvacrol could target the SARS-Cov-2 spike protein of the Omicron variant RBD domain. Molecular dynamics simulations disclosed that these compounds have a stable dynamic behavior over 100 ns as compared to remdesivir. Chemo-computational toxicity prediction using Protox II webserver indicated that thymol and carvacrol could be safely and effectively used as drug candidates to tackle bacterial, fungal, and viral infections as compared to chemical medication.

Intraspecific divergence in essential oil content, composition and genes expression patterns of monoterpene synthesis in Origanum vulgare subsp. vulgare and subsp. gracile under salinity stress.

BACKGROUND: Oregano (Origanum vulgare L.), one of the important medicinal plants in the world, has valuable pharmacological compounds with antimicrobial, antiviral, antioxidant, anti-inflammatory, antispasmodic, antiurolithic, antiproliferative and neuroprotective activities. Phenolic monoterpenes such as thymol and carvacrol with many medical importance are found in Oregano essential oil. The biosynthesis of these compounds is carried out through the methyl erythritol-4 phosphate (MEP) pathway. Environmental stresses such as salinity might improve the secondary metabolites in medicinal plants. The influence of salinity stress (0 (control), 25, 50 and 100 mM NaCl) on the essential oil content, composition and expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), γ-terpinene synthase (Ovtps2) and cytochrome P450 monooxygenases (CYP71D180) genes involved in thymol and carvacrol biosynthesis, was investigated in two oregano subspecies (vulgare and gracile). RESULTS: Essential oil content was increased at low NaCl concentration (25 mM) compared with non-stress conditions, whereas it was decreased as salinity stress intensified (50 and 100 mM). Essential oil content was significantly higher in subsp. gracile than subsp. vulgare. The highest (0.20 mL pot-1) and lowest (0.06 mL pot-1) amount of essential oil yield was obtained in subsp. gracile at 25 and 100 mM NaCl, respectively. The content of carvacrol, as the main component of essential oil, decreased with increasing salinity level in subsp. gracile, but increased in subsp. vulgare. The highest expression of DXR, Ovtps2 and CYP71D180 genes was observed at 50 mM NaCl in subsp. vulgare. While, in subsp. gracile, the expression of the mentioned genes decreased with increasing salinity levels. A positive correlation was obtained between the expression of DXR, Ovtps2 and CYP71D180 genes with carvacrol content in both subspecies. On the other hand, a negative correlation was found between the expression of CYP71D180 and carvacrol content in subsp. gracile. CONCLUSIONS: The findings of this study demonstrated that both oregano subspecies can tolerate NaCl salinity up to 50 mM without significant reduction in essential oil yield. Also, moderate salinity stress (50 mM NaCl) in subsp. vulgare might increase the carvacrol content partly via increment the expression levels of DXR, Ovtps2 and CYP71D180 genes.

Single-cell multi-omics in the medicinal plant Catharanthus roseus.

Advances in omics technologies now permit the generation of highly contiguous genome assemblies, detection of transcripts and metabolites at the level of single cells and high-resolution determination of gene regulatory features. Here, using a complementary, multi-omics approach, we interrogated the monoterpene indole alkaloid (MIA) biosynthetic pathway in Catharanthus roseus, a source of leading anticancer drugs. We identified clusters of genes involved in MIA biosynthesis on the eight C. roseus chromosomes and extensive gene duplication of MIA pathway genes. Clustering was not limited to the linear genome, and through chromatin interaction data, MIA pathway genes were present within the same topologically associated domain, permitting the identification of a secologanin transporter. Single-cell RNA-sequencing revealed sequential cell-type-specific partitioning of the leaf MIA biosynthetic pathway that, when coupled with a single-cell metabolomics approach, permitted the identification of a reductase that yields the bis-indole alkaloid anhydrovinblastine. We also revealed cell-type-specific expression in the root MIA pathway.

Characterization of Cinnamomum kanehirae Extract-Stimulated Triterpenoids Synthesis in Submerged Fermentation of Antrodia camphorata via Untargeted Metabolomics.

The underlying mechanisms of Cinnamomum kanehirae-stimulated growth and metabolism of Antrodia camphorata remain unknown. Herein, we first observed that the methanol extract of C. kanehirae trunk (MECK) (2 g/L) showed a potent stimulatory effect on A. camphorata triterpenoids production (115.6 mg/L). Second, MECK treatment considerably increased the category and abundance of many secondary metabolites in the mycelia. We identified 93 terpenoids (8 newly formed and 49 upregulated) in the MECK-treated mycelia, wherein 21 terpenoids were the same as those in the fruiting bodies. Third, 42 out of the 93 terpenoids were annotated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, mainly involving monoterpenoids and diterpenoids syntheses. Finally, 27 monoterpenes and 16 sesquiterpenes were detected in the MECK, and the two terpenoids with the highest abundance (linalool and α-pinene) were selected for verification and found to considerably increase the terpenoids production of A. camphorata and demonstrate the regulation of mRNA expression levels of nine key genes in the mevalonate pathway via RT-qPCR. This study is beneficial for elucidating the terpenoids synthesis mechanism in A. camphorata.

Terpenoid VOC profiles and functional characterization of terpene synthases in diploid and tetraploid cytotypes of Chrysanthemum indicum L.

Chrysanthemum indicum L. is a valuable medicinal plant with diploid and tetraploid forms that are widely distributed in central and southern China, and it contains abundant volatile organic compounds (VOCs). Despite the discovery of some terpene synthase (TPS) in C. indicum (i.e., CiTPS) in previous studies, many TPSs and their corresponding terpene biosynthesis pathways have yet to be discovered. In the present study, terpenoid VOCs in different tissues from two cytotypes of C. indicum were analyzed. We identified 52 types of terpenoid VOCs and systematically investigated the content and distribution of these compounds in various tissues. The two cytotypes of C. indicum exhibited different volatile terpenoid profiles. The content of monoterpenes and sesquiterpenes in the two cytotypes showed an opposite trend. In addition, four full-length candidate TPSs (named CiTPS5-8) were cloned from Ci-GD4x, and their homologous TPS genes were screened based on the genome data of Ci-HB2x. These eight TPSs displayed various tissue expression patterns and were discovered to produce 22 terpenoids, 5 of which are monoterpenes and 17 are sesquiterpenes. We further proposed corresponding terpene synthesis pathways, which can enable the establishment of an understanding of the volatile terpenoid profiles of C. indicum with different cytotypes. This knowledge may provide a further understanding of germplasm in C. indicum and may be useful for biotechnology applications of Chrysanthemum plants.

Gene coexpression networks allow the discovery of two strictosidine synthases underlying monoterpene indole alkaloid biosynthesis in Uncaria rhynchophylla.

Plant-derived monoterpene indole alkaloids (MIAs) from Uncaria rhynchophylla (UR) have huge medicinal properties in treating Alzheimer's disease, Parkinson's disease, and depression. Although many bioactive UR-MIA products have been isolated as drugs, their biosynthetic pathway remains largely unexplored. In this study, untargeted metabolome identified 79 MIA features in UR tissues (leaf, branch stem, hook stem, and stem), of which 30 MIAs were differentially accumulated among different tissues. Short time series expression analysis captured 58 pathway genes and 12 hub regulators responsible for UR-MIA biosynthesis and regulation, which were strong links with main UR-MIA features. Coexpression networks further pointed to two strictosidine synthases (UrSTR1/5) that were coregulated with multiple MIA-related genes and highly correlated with UR-MIA features (r > 0.7, P < 0.005). Both UrSTR1/5 catalyzed the formation of strictosidine with tryptamine and secologanin as substrates, highlighting the importance of key residues (UrSTR1: Glu309, Tyr155; UrSTR5: Glu295, Tyr141). Further, overexpression of UrSTR1/5 in UR hairy roots constitutively increased the biosynthesis of bioactive UR-MIAs (rhynchophylline, isorhynchophylline, corynoxeine, etc), whereas RNAi of UrSTR1/5 significantly decreased UR-MIA biosynthesis. Collectively, our work not only provides candidates for reconstituting the biosynthesis of bioactive UR-MIAs in heterologous hosts but also highlights a powerful strategy for mining natural product biosynthesis in medicinal plants.

[Cloning of StHD1 and StHD8 from Schizonepeta tenuifolia and function of regulating glandular trichome development].

Hd-Zip, a unique transcription factor in plant kingdom, influences the growth, development, and secondary metabolism of plants. Hd-zip Ⅳ is thought to play an important role in trichome development of Schizonepeta tenuifolia. This study aims to explore the functions of StHD1 and StHD8 in Hd-zip Ⅳ subfamily in peltate glandular trichome development. To be specific, the expression patterns of the two genes and interaction between the proteins encoded by them were analyzed based on transcriptome sequencing and two-hybrid screening. The subcellular localization was performed and functions of the genes were verified in tobacco and S. tenuifolia. The results showed that StHD1 and StHD8 had high similarity to HD-Zip Ⅳ proteins of other plants and they all had the characteristic conserved domains of HD-Zip Ⅳ subfamily. They were located in the nucleus. The two genes mainly expressed in young tissues and spikes, and StHD1 and StHD8 proteins interacted with each other. The density and length of glandular trichomes increased significantly in tobacco plants with the overexpression of StHD1 and StHD8. Inhibiting the expression of StHD1 and StHD8 by VIGS(virus-induced gene silencing) in S. tenuifolia resulted in the reduction in the density of peltate glandular trichomes, the expression of key genes related to mono-terpene synthesis, and the relative content of limonene and pulegone, the main components of monoterpene. These results suggested that StHD1 and StHD8 of S. tenuifolia formed a complex to regulate glandular trichomes and affect the biosynthesis of monoterpenes.

Full-Length Transcriptomic Sequencing and Temporal Transcriptome Expression Profiling Analyses Offer Insights into Terpenoid Biosynthesis in Artemisia argyi.

Artemisiae argyi Folium is a traditional herbal medicine used for moxibustion heat therapy in China. The volatile oils in A.argyi leaves are closely related to its medicinal value. Records suggest that the levels of these terpenoids components within the leaves vary as a function of harvest time, with June being the optimal time for A. argyi harvesting, owing to the high levels of active ingredients during this month. However, the molecular mechanisms governing terpenoid biosynthesis and the time-dependent changes in this activity remain unclear. In this study, GC-MS analysis revealed that volatile oil levels varied across four different harvest months (April, May, June, and July) in A. argyi leaves, and the primarily terpenoids components (including both monoterpenes and sesquiterpenes) reached peak levels in early June. Through single-molecule real-time (SMRT) sequencing, corrected by Illumina RNA-sequencing (RNA-Seq), 44 full-length transcripts potentially involved in terpenoid biosynthesis were identified in this study. Differentially expressed genes (DEGs) exhibiting time-dependent expression patterns were divided into 12 coexpression clusters. Integrated chemical and transcriptomic analyses revealed distinct time-specific transcriptomic patterns associated with terpenoid biosynthesis. Subsequent hierarchical clustering and correlation analyses ultimately identified six transcripts that were closely linked to the production of these two types of terpenoid within A. argyi leaves, revealing that the structural diversity of terpenoid is related to the generation of the diverse terpene skeletons by prenyltransferase (TPS) family of enzymes. These findings can guide further studies of the molecular mechanisms underlying the quality of A. argyi leaves, aiding in the selection of optimal timing for harvests of A. argyi.

[Identification of genes involved in biosynthesis of paeoniflorin in Paeonia lactiflora based on transcriptome analysis].

Paeoniflorin, a representative pinane monoterpene glycoside, is the main active component and quality index of Paeoniae Radix Alba and Paeoniae Radix Rubra.The possible biosynthesis of paeoniflorin is as follows: GPP is derived from mevalonate(MVA) and/or 2-C-methyl-D-erythritol 4-phosphate(MEP) pathway(s) followed by the catalysis with terpene synthase, cytochrome P450(CYP450), UDP-glucuronosyltransferase(UGT), and acyltransferase(AT), respectively.This study aims to explore the genes rela-ted to the biosynthesis of paeoniflorin.To be specific, the cDNA libraries for flowers, leaves, and roots of Paeonia lactiflora were established and sequenced.A total of 30 609 open reading frames(ORFs) were yielded.Through functional annotation and expression analysis of all CYP450 genes in the transcriptome, 11 CYP450 genes belonging to CYP71 A and CYP71 D subfamilies and showing expression trend consistent with monoterpene synthase PlPIN that may be involved in paeoniflorin biosynthesis were screened out.Subsequently, 7 UGT genes and 9 AT genes demonstrating the expression trend consistent with PlPIN which were possibly involved in paeoniflorin biosynthesis were further screened by functional annotation analysis, full-length sequence analysis, expression analysis, and phylogeny analysis.This study provided a systematic screening method with smaller number of candidate genes, thus reducing the workload of functional gene verification.The result laid a foundation for analyzing the biosynthesis pathway of paeoniflorin and the formation mechanism.

Toxicity of Selected Monoterpenes and Essential Oils Rich in These Compounds.

Monoterpenes make up the largest group of plant secondary metabolites. They can be found in numerous plants, among others, the Lamiaceae family. The compounds demonstrate antioxidative, antibacterial, sedative and anti-inflammatory activity, hence, they are often employed in medicine and pharmaceuticals. Additionally, their fragrant character is often made use of, notably in the food and cosmetic industries. Nevertheless, long-lasting studies have revealed their toxic properties. This fact has led to a detailed analysis of the compounds towards their side effects on the human organism. Although most are safe for human food and medical applications, there are monoterpene compounds that, in certain amounts or under particular circumstances (e.g., pregnancy), can cause serious disorders. The presented review characterises in vitro and in vivo, the toxic character of selected monoterpenes (α-terpinene, camphor, citral, limonene, pulegone, thujone), as well as that of their original plant sources and their essential oils. The selected monoterpenes reveal various toxic properties among which are embryotoxic, neurotoxic, allergenic and genotoxic. It is also known that the essential oils of popular plants can also reveal toxic characteristics that many people are unaware of.

[Dynamic control of ERG20 expression to improve production of monoterpenes by engineering Saccharomyces cerevisiae].

Monoterpenes are widely used in cosmetics, food, medicine, agriculture and other fields. With the development of synthetic biology, it is considered as a potential way to create microbial cell factories to produce monoterpenes. Engineering Saccharomyces cerevisiae to produce monoterpenes has been a research hotspot in synthetic biology. In S. cerevisiae, the production of geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) is catalyzed by a bifunctional enzyme farnesyl pyrophosphate synthetase(encoded by ERG20 gene) which is inclined to synthesize FPP essential for yeast growth. Therefore, reasonable control of FPP synthesis is the basis for efficient monoterpene synthesis in yeast cell factories. In order to achieve dynamic control from GPP to FPP biosynthesis in S. cerevisiae, we obtained a novel chassis strain HP001-pERG1-ERG20 by replacing the ERG20 promoter of the chassis strain HP001 with the promoter of cyclosqualene cyclase(ERG1) gene. Further, we reconstructed the metabolic pathway by using GPP and neryl diphosphate(NPP), cis-GPP as substrates in HP001-pERG1-ERG20. The yield of GPP-derived linalool increased by 42.5% to 7.6 mg·L~(-1), and that of NPP-derived nerol increased by 1 436.4% to 8.3 mg·L~(-1). This study provides a basis for the production of monoterpenes by microbial fermentation.

Engineering Catharanthus roseus monoterpenoid indole alkaloid pathway in yeast.

Catharanthus roseus (Madagascar periwinkle), a medicinal plant possessing high pharmacological attributes, is widely recognized for the biosynthesis of anticancer monoterpenoid indole alkaloids (MIAs) - vinblastine and vincristine. The plant is known to biosynthesize more than 130 different bioactive MIAs, highly acclaimed in traditional and modern medicinal therapies. The MIA biosynthesis is strictly regulated at developmental and spatial-temporal stages and requires a well-defined cellular and sub-cellular compartmentation for completion of the entire MIAs biosynthesis. However, due to their cytotoxic nature, the production of vinblastine and vincristine occurs in low concentrations in planta and the absence of chemical synthesis alternatives projects a huge gap in demand and supply, leading to high market price. With research investigations spanning more than four decades, plant tissue culture and metabolic engineering (ME)-based studies were attempted to explore, understand, explain, improve and enhance the MIA biosynthesis using homologous and heterologous systems. Presently, metabolic engineering and synthetic biology are the two powerful tools that are contributing majorly in elucidating MIA biosynthesis. This review concentrates mainly on the efforts made through metabolic engineering of MIAs in heterologous microbial factories. KEY POINTS: • Yeast engineering provides alternative production source of phytomolecules • Yeast engineering also helps to discover missing plant pathway enzymes and genes.

Thymol and carvacrol supplementation in poultry health and performance.

BACKGROUND: Thymol and carvacrol as natural essential oils and phenol compounds are components derived from some medicinal plants, such as thyme and oregano species. OBJECTIVES: The increasing demands in organic and healthy meat and egg consumption in human society have made it necessary to consider alternative natural compounds for the replacement of chemical compounds in poultry production. The chemical compounds can remain in meat and eggs and cause complications in human health. Therefore, these natural compounds can be fed with a higher safety in poultry production with specific effects. In this regard, the role of thymol and carvacrol as natural compounds in the poultry production has been discussed in the review. METHODS: In this study, by searching for keywords related to thymol and carvacrol in poultry production in Google Scholar database, the articles related to different aspects of the biological effects of these two phytogenes in poultry production were selected and analyzed. RESULTS: A review of previous studies has shown that thymol and carvacrol possess a wide range of biological activities, including antibacterial, antiviral, antioxidant, anti-inflammatory, modulating of immunity response and regulating of the gut microbial population. Also, in meat type chickens can promote growth and influence feed utilization. The beneficial effect of this compound was evaluated in hepatic toxicity and demonstrated as a hepatoprotective compound in chickens. Furthermore, these compounds can affect the behavior of layers and influence egg composition, eggshell thickness, and the sensory quality of eggs. CONCLUSION: It seems that with the increasing demand for healthy protein products, these compounds can be used to improve performance as a substitute alternative for chemical compounds in healthy poultry farms.

[Effect of hot or warm property on skin toxicity of essential oil as penetration enhancer and its mechanism].

To compare the effect of hot or warm property of Chinese medicine(CM) on the skin toxicity of essential oils(EOs) as penetration enhancer in vitro and in vivo, and explore the mechanism. EOs were extracted from WIM of Bichengqie(Litseae Fructus), Dingxiang(Flos Syzygii Aromatici), Huajiao(Pericarpium Zanthoxyli Bungeani), and Xiaohuixiang(Fructus Foeniculi) with warm property, and Ganjiang(Rhizoma Zingiberis), Gaoliangjiang(Rhizoma Alpiniae Officinari), Hujiao(Fructus Piperis), and Wuzhuyu(Fructus Evodiae Rutaecarpae) with hot property, respectively. Then the in vitro toxicity was evaluated by human keratinocyte cytotoxicity. In vivo skin irritation potency was also evaluated through pathological observation after topical administration. The components, especially those located in stratum corneum, were analyzed by GC-MS. The main components, namely monoterpenes and sesquiterpenes, of EOs extracted from CM with hot property,were detected for the interaction with keratino-lipid ceramide 3 by molecular simulation technology; and the interaction energy value was calculated based on the optimal conformation. It was found that the skin cell toxicity of EOs from CM with hot property was significantly higher than that of EOs from CM with warm property. However, there was no significant difference between them by in vivo skin irritation evaluation. Whether from CM with hot property or warm property, EOs showed a significant reduced toxicity compared with azone. Sesquiterpenes(33.56%±19.38%) were found to be one of the main components in EOs from CM with hot property, while almost no sesquiterpenes was found in EOs from CM with warm property. After topical administration of EOs from CM with hot property, sesquiterpenes were demonstrated to be prone to locate in stratum corneum. The results of molecular simulation also revealed that the interaction between sesquiterpenes and ceramide 3 was significantly stronger than that of monoterpenes(P<0.01). In conclusion, the location of sesquiterpenes in stratum corneum resulted in the significant difference between in vitro skin cell toxicity and in vivo skin irritation potency. The EOs from CM with hot property shall be taken into account for further development of potent penetration enhancer.

Physiological and biochemical responses of green and red perilla to LED-based light.

BACKGROUND: Light-emitting diodes (LEDs) are widely used in closed-type plant production systems to improve biomass and accumulate bioactive compounds in plants. Perilla has been commonly used as herbal medicine because of its health-promoting effects. This study aimed to investigate the physiological and biochemical responses of green and red perilla under various visible-light spectra. RESULTS: Results showed that red (R) LEDs improved fresh weights of shoots and roots, plant height, internode length, node number and leaf area, as well as photosynthetic rate of green and red perilla plants compared to blue (B) LEDs and RB combined LEDs. Meanwhile, B resulted in higher stomatal conductance, transpiration rate and Fv/Fm compared to R. Supplementation of green (G) and far-red (FR) did not enhance perilla growth. Reduction or absence of B decreased leaf thickness, adaxial and abaxial epidermis, and palisade and spongy mesophyll. Total phenolic content, antioxidant capacity, rosmarinic acid content and caffeic acid content of green perilla were higher under R, R8B2 and RGB + FR, while greater values were obtained in red perilla under R. Accumulation of perillaldehyde, luteolin and apigenin presented different trends from those of rosmarinic and caffeic acids in both cultivars. CONCLUSIONS: Growth and accumulation of bioactive compounds in green perilla were greater than in red perilla under similar light quality, and R LEDs or a higher R ratio in combination treatments were suitable for cultivating high-quality green and red perilla plants in closed-type plant factories. © 2020 Society of Chemical Industry.

Secondary Metabolic Profile as a Tool for Distinction and Characterization of Cultivars of Black Pepper (Piper nigrum L.) Cultivated in Pará State, Brazil.

This study evaluated the chemical compositions of the leaves and fruits of eight black pepper cultivars cultivated in Pará State (Amazon, Brazil). Hydrodistillation and gas chromatography-mass spectrometry were employed to extract and analyze the volatile compounds, respectively. Sesquiterpene hydrocarbons were predominant (58.5-90.9%) in the cultivars "Cingapura", "Equador", "Guajarina", "Iaçará", and "Kottanadan", and "Bragantina", "Clonada", and "Uthirankota" displayed oxygenated sesquiterpenoids (50.6-75.0%). The multivariate statistical analysis applied using volatile composition grouped the samples into four groups: γ-Elemene, curzerene, and δ-elemene ("Equador"/"Guajarina", I); δ-elemene ("Iaçará"/"Kottanadan"/"Cingapura", II); elemol ("Clonada"/"Uthirankota", III) and α-muurolol, bicyclogermacrene, and cubebol ("Bragantina", IV). The major compounds in all fruit samples were monoterpene hydrocarbons such as α-pinene, ß-pinene, and limonene. Among the cultivar leaves, phenolics content (44.75-140.53 mg GAE·g-1 FW), the enzymatic activity of phenylalanine-ammonia lyase (20.19-57.22 µU·mL-1), and carotenoids (0.21-2.31 µg·mL-1) displayed significant variations. Due to black pepper's susceptibility to Fusarium infection, a molecular docking analysis was carried out on Fusarium protein targets using each cultivar's volatile components. F. oxysporum endoglucanase was identified as the preferential protein target of the compounds. These results can be used to identify chemical markers related to the susceptibility degree of black pepper cultivars to plant diseases prevalent in Pará State.

The permeability characteristics and interaction of main components from Si-Ni-San in a MDCK epithelial cell monolayer model.

1. Si-Ni-San (SNS) possesses extensive therapeutic effects, however, the extent to which main components are absorbed and the mechanisms involved are controversial. 2. In this study, MDCK cell model was used to determine the permeability characteristics and interaction between the major components of Si-Ni-San, including saikosaponin a, paeoniflorin, naringin and glycyrrhizic acid. 3. The transport of the major components was concentration-dependent in both directions. Moreover, the transport of paeoniflorin, naringin and glycyrrhizic acid was significantly reduced at 4 °C or in the presence of NaN3. Additionally, the efflux of paeoniflorin and naringin were apparently reduced in the presence of P-gp inhibitor verapamil. The transport of glycyrrhizic acid was clearly inhibited by the inhibitors of MRP2, indicating that MRP2 may be involved in the transport of glycyrrhizic acid. However, the results indicated that saikosaponin a was absorbed mainly by passive diffusion. Furthermore, the combined incubation of four major components had a powerful sorbefacient effect than a single drug used alone which may be regulated by tight junctions. 4. Taken together, our study provides useful information for pharmacological applications of Si-Ni-San and offers new insights into this ancient decoction for further researches, especially in drug synergism.

Developmental Methylome of the Medicinal Plant Catharanthus roseus Unravels the Tissue-Specific Control of the Monoterpene Indole Alkaloid Pathway by DNA Methylation.

Catharanthus roseus produces a wide spectrum of monoterpene indole alkaloids (MIAs). MIA biosynthesis requires a tightly coordinated pathway involving more than 30 enzymatic steps that are spatio-temporally and environmentally regulated so that some MIAs specifically accumulate in restricted plant parts. The first regulatory layer involves a complex network of transcription factors from the basic Helix Loop Helix (bHLH) or AP2 families. In the present manuscript, we investigated whether an additional epigenetic layer could control the organ-, developmental- and environmental-specificity of MIA accumulation. We used Whole-Genome Bisulfite Sequencing (WGBS) together with RNA-seq to identify differentially methylated and expressed genes among nine samples reflecting different plant organs and experimental conditions. Tissue specific gene expression was associated with specific methylation signatures depending on cytosine contexts and gene parts. Some genes encoding key enzymatic steps from the MIA pathway were found to be simultaneously differentially expressed and methylated in agreement with the corresponding MIA accumulation. In addition, we found that transcription factors were strikingly concerned by DNA methylation variations. Altogether, our integrative analysis supports an epigenetic regulation of specialized metabolisms in plants and more likely targeting transcription factors which in turn may control the expression of enzyme-encoding genes.