Influence of Genista Tinctoria L or Methylparaben on Subchronic Toxicity of Bisphenol A in Rats

Objective To evaluate the influence of an extract of Genista tinctoria L. herba （GT） or methylparaben （MP） on histopathological changes and 2 biomarkers of oxidative stress in rats subchronicly exposed to bisphenol A （BPA）. Methods Adult female Wistar rats were orally exposed for 90 d to BPA （50 mg/kg）, BPA＋GT （35 mg isoflavones/kg） or BPA＋MP （250 mg/kg）. Plasma and tissue samples were taken from liver, kidney, thyroid, uterus, ovary, and mammary gland after 30, 60, and 90 d of exposure respectively. Lipid peroxidation and in vivo hydroxyl radical production were evaluated by histological analysis along with malondialdehyde and 2,3-dihydroxybenzoic acid detection. Results The severity of histopathological changes in liver and kidneys was lower after GT treatment than after BPA or BPA＋MP treatment. A minimal thyroid receptor antagonist effect was only observed after BPA＋MP treatment. The abnormal folliculogenesis increased in a time-dependent manner, and the number of corpus luteum decreased. No significant histological alterations were found in the uterus. The mammary gland displayed specific estrogen stimulation changes at all periods. Both MP and GT revealed antioxidant properties reducing lipid peroxidation and BPA-induced hydroxyl radical generation. Conclusion GT L. extract ameliorates the toxic effects of BPA and is proved to have antioxidant potential and antitoxic effect. MP has antioxidant properties, but has either no effect or exacerbates the BPA-induced histopathological changes.

Biological activities of the Ornithogalum orthophyllum and its in silico ADMET profile

Background:The bulbs and aerial parts of Ornithogalum are used in Turkey both as food and to treat various ailments,and some of its medical applications are well known.However,the biological activities of Ornithogalum orthophyllum have not yet been investigated.The objective of this study was to examine the antioxidant,urease and cholinesterase enzyme inhibition,and cytotoxic activities of different extracts obtained from the bulb and aerial parts of the O.orthophyllum plant.In addition,the absorption,distribution,metabolism,excretion,and toxicity properties of some phenolic compounds in plants were examined in silico.Methods:The antioxidant activity of the extracts obtained from the aerial parts of O.orthophyllum was investigated using the 2,2-diphenyl-1-picrylhydrazyl,2,2-azinobis(3-ethylbenzothiazoline-6-sulphonic acid),ferric reducing antioxidant power,and cupric reducing antioxidant capacity methods.Urease and cholinesterase enzyme inhibition were determined by using the indophenol and Ellman methods,respectively.The cytotoxic activity of the extracts was measured using a test based on the luminometric readings of the adenosine triphosphate levels of the cells.pkCSM,a free online web server(http://structure.bioc.cam.ac.uk/pkcsm)was used to predict the properties of the compounds analyzed.Results:The methanol extract of the aerial parts of O.orthophyllum was observed to have strong antioxidant and acetylcholinesterase enzyme inhibition potential.The petroleum ether extract of the aerial parts showed the highest anti-urease activity.The chloroform extract of the aerial parts exhibited the highest cytotoxic effect against A431 human epidermoid carcinoma cells.The absorption percentages of protocatechuic acid,p-hydroxybenzoic acid,vanillic acid,and p-coumaric acid compounds from the small intestine were between 71.17%and 93.49%,which were quite high.All the compounds were predicted to be unable to penetrate the central nervous system due to their inability to cross the blood-brain barrier.Not all compounds analyzed were predicted to have mutagenic,hepatotoxic,or minnow toxicity effects.Conclusion:The extracts obtained from the aerial parts of O.orthophyllum have strong biological activity and contain compounds that are well-absorbed and do not have mutagenic,hepatotoxic,or minnow toxicity effects,suggesting that they can be used as natural medicinal and nutritional sources in the future.

The ERF transcription factor LTF1 activates DIR1 to control stereoselective synthesis of antiviral lignans and stress defense in Isatis indigotica roots

Lignans are a powerful weapon for plants to resist stresses and have diverse bioactive functions to protect human health.Elucidating the mechanisms of stereoselective biosynthesis and response to stresses of lignans is important for the guidance of plant improvement.Here,we identified the complete pathway to stereoselectively synthesize antiviral(-)-lariciresinol glucosides in Isatis indigotica roots,which consists of three-step sequential stereoselective enzymes DIR1/2,PLR,and UGT71B2.DIR1 was further identified as the key gene in respoJanuary 2024nse to stresses and was able to trigger stress defenses by mediating the elevation in lignan content.Mechanistically,the phytohormone-responsive ERF transcription factor LTF1 colocalized with DIR1 in the cell periphery of the vascular regions in mature roots and helped resist biotic and abiotic stresses by directly regulating the expression of DIR1.These systematic results suggest that DIR1 as the first common step of the lignan pathway cooperates with PLR and UGT71B2 to stereoselectively synthesize(-)-lariciresinol derived antiviral lignans in I.indigotica roots and is also a part of the LTF1-mediated regulatory network to resist stresses.In conclusion,the LTF1-DIR1 module is an ideal engineering target to improve plant Defenses while increasing the content of valuable lignans in plants.

Molecular regulation of the key specialized metabolism pathways in medicinal plants

The basis of modern pharmacology is the human ability to exploit the production of specialized metabolites from medical plants,for example,terpenoids,alkaloids,and phenolic acids.However,in most cases,the availability of these valuable compounds is limited by cellular or organelle barriers or spatio-temporal accumulation patterns within different plant tissues.Transcription factors(TFs)regulate biosynthesis of these specialized metabolites by tightly controlling the expression of biosynthetic genes.Cutting-edge technologies and/or combining multiple strategies and approaches have been applied to elucidate the role of TFs.In this review,we focus on recent progress in the transcription regulation mechanism of representative high-value products and describe the transcriptional regulatory network,and future perspectives are discussed,which will help develop high-yield plant resources.

Total synthesis of lissodendoric acid A by[4+2]cycloaddition of transient cyclic allene

Lissodendoric acid A is a member of manzamine alkaloid family[1],which is a popular target for total synthesis due to its complex structure and promising biological activity[2].Lissodendoric acid A was isolated from the marine sponge Lissodendoryx florida in 2017 by Stonik’s group.Related research has demonstrated that this natural product exerted therapeutic effect on a Parkinson’s disease model containing Neuro 2a cells and somewhat increased the survival of these cells upon treatment with dopamine[3].The core skeleton of lissodendoric acid A features an azadecalin scaffold containing a conjugated diene,a carboxylic acid substituent and two stereocenters,which poses a forbidding challenge on total synthesis.Since the 1960s,the small ring structure bearing allene has been known to chemists as an unconventional transient intermediate[4,5],whose unique chemical structure makes it highly reactive,allowing events such as cycloaddition,nucleophilic addition or metal catalysis to occur.However,the application of cyclic allenes in total synthesis of complex natural products has rarely been reported compared with their close relatives,such as arynes and strained cyclic alkynes[6].

Genome-wide analysis of AP2/ERF superfamily in Isatis indigotica

Objective AP2/ERF(APETALA2/ethylene-responsive factor)superfamily is one of the largest gene families in plants and has been reported to participate in various biological processes,such as the regulation of biosynthesis of active lignan.However,few studies have investigated the genome-wide role of the AP2/ERF superfamily in Isatis indigotica.This study establishes a complete picture of the AP2/ERF superfamily in I.indigotica and contributes valuable information for further functional characterization of IiAP2/ERF genes and supports further metabolic engineering.Methods To identify the IiAP2/ERF superfamily genes,the AP2/ERF sequences from Arabidopsis thaliana and Brassica rapa were used as query sequences in the basic local alignment search tool.Bioinformatic analyses were conducted to investigate the protein structure,motif composition,chromosome location,phylogenetic relationship,and interaction network of the IiAP2/ERF superfamily genes.The accuracy of omics data was verified by quantitative polymerase chain reaction and heatmap analyses.Results One hundred and twenty-six putative IiAP2/ERF genes in total were identified from the I.indigotica genome database in this study.By sequence alignment and phylogenetic analysis,the IiAP2/ERF genes were classified into 5 groups including AP2,ERF,DREB(dehydration-responsive element-binding factor),Soloist and RAV(related to abscisic acid insensitive 3/viviparous 1)subfamilies.Among which,122 members were unevenly distributed across seven chromosomes.Sequence alignment showed that I.indigotica and A.thaliana had 30 pairs of orthologous genes,and we constructed their interaction network.The comprehensive analysis of gene expression pattern in different tissues suggested that these genes may play a significant role in organ growth and development of I.indigotica.Members that may regulate lignan biosynthesis in roots were also preliminarily identified.Ribonucleic acid sequencing analysis revealed that the expression of 76 IiAP2/ERF genes were up-or down-regulated under salt or drought treatment,among which,33 IiAP2/ERF genes were regulated by both stresses.Conclusion This study undertook a genome-wide characterization of the AP2/ERF superfamily in I.indigotica,providing valuable information for further functional characterization of IiAP2/ERF genes and discovery of genetic targets for metabolic engineering.

Ribonucleotide reductase M2 (RRM2): Regulation, function and targeting strategy in human cancer

Ribonucleotide reductase M2(RRM2)is a small subunit in ribonucleotide reduc-tases,which participate in nucleotide metabolism and catalyze the conversion of nucleotides to deoxynucleotides,maintaining the dNTP pools for DNA biosynthesis,repair,and replication.RRM2 performs a critical role in the malignant biological behaviors of cancers.The structure,regulation,and function of RRM2 and its inhibitors were discussed.RRM2 gene can produce two transcripts encoding the same ORF.RRM2 expression is regulated at multiple levels during the processes from transcription to translation.Moreover,this gene is associated with resistance,regulated cell death,and tumor immunity.In order to develop and design inhibitors of RRM2,appropriate strategies can be adopted based on different mechanisms.Thus,a greater appreciation of the characteristics of RRM2 is a benefit for understanding tumorigenesis,resistance in cancer,and tumor microenvironment.Moreover,RRM2-targeted therapy will be more attention in future therapeutic approaches for enhancement of treatment effects and amelioration of the dismal prognosis.

Artemisia annua glandular secretory trichomes: the biofactory of antimalarial agent artemisinin

Artemisinin, the key ingredient of first-line antimalarial drugs, has large demand every year. The native plant, which produces small quantities of artemisinin, remains as its main source and thus results in a short supply of artemisinin. Intensified efforts have been carried out to elevate artemisinin production. However, the routine metabolic engineering strategy, via overexpressing or down-regulating genes in artemisinin biosynthesis branch pathways, was not very effective as desired. Glandular secretory trichomes, sites of artemisinin biosynthesis on the surface of Artemisia annua L.(A. annua), are the new target for increasing artemisinin yield. In general, the population and morphology of glandular secretory trichomes in A. annua(Aa GSTs) are often positively correlated with artemisinin content. Improved understanding of Aa GSTs will shed light on the opportunities for increasing plant-derived artemisinin. This review article will refresh classification of trichomes in A. annua and provide an overview of the recent achievements regarding Aa GSTs and artemisinin. To have a full understanding of Aa GSTs,factors that are associated with trichome morphology and density will have to be further investigated, such as genes,micro RNAs and phytohormones. The purpose of thisreview was to(1) update the knowledge of the relation between Aa GSTs and artemisinin, and(2) propose new avenues to increase artemisinin yield by harnessing the potential biofactories, Aa GSTs.

Gas chromatography-mass spectrometry analysis of essential oils from five parts of Chaihu(Radix Bupleuri Chinensis)

OBJECTIVE: To analyze the essential oils from flowers, leaves, stems, roots, and fruits of Chaihu(Radix Bupleuri Chinensis).METHODS: We extracted essential oils from different parts of Chaihu(Radix Bupleuri Chinensis) using a steam distillation method. The essential oils were analyzed by gas chromatography-mass spectrometry(GC-MS). Data were collected in full scan mode(m/z 60-600). Volatile components were identified based on their retention indices and by comparing their mass spectra with those in the National Institute of Standards and Technology 2005 database assisted by tandem mass spectrometry information. The relative content of each constituent wasdetermined by area normalization.RESULTS: We identified 111 components, of which12 were common to all 5 parts, 30 were found only in roots, 14 were found only in flowers, 6 were found only in leaves, 4 were found only in stems,and 17 were found only in fruits.CONCLUSION: Our results show that the stems,flowers, leaves, and fruits of Chaihu(Radix Bupleuri Chinensis)contain a high concentration of essential oils, and that the exact composition of the essential oils differs among the plant parts. To develop new medicines and make full use of the Chaihu(Radix Bupleuri Chinensis)resource, it is important to characterize the essential oils from different parts of the plant. In future research, it will be important to determine the pharmacological effects of the various components and the essential oil mixtures.

Computational identification and systematic classification of novel GRAS genes in Isatis indigotica

Isatis indigotica Fort., belonging to Cruciferae, is one of the most commonly used plants in traditional Chinese medicine. The accumulation of the effective components of I. indigotica is related with its growth conditions. The GRAS genes are members of a multigene family of transcriptional regulators that play a crucial role in plant growth. Although the activities of many GRAS genes have long been recognized, only in recent years were some of them identified and functionally characterized in detail. In the present study, 41 GRAS genes were identified from I. indigotica through bioinformatics methods for the first time. They were classified into ten groups according to the classification of Arabidopsis and rice. The characterization, gene structure, conserved motifs, disordered N-terminal domains, and phylogenetic reconstruction of these GRASs were analyzed. Forty-three orthologous gene pairs were shared by I. indigotica and Arabidopsis, and interaction networks of these orthologous genes were constructed. Furthermore, gene expression patterns were investigated by analysis in methyl jasmonate(Me JA)-treated I. indigotica hairy roots based on RNA-seq data. In conclusion, this comprehensive analysis would provide rich resources for further studies of GRAS protein functions in this plant.

HPPR encodes the hydroxyphenylpyruvate reductase required for the biosynthesis of hydrophilic phenolic acids in Salvia miltiorrhiza

Salvia miltiorrhiza is a medicinal plant widely used in the treatment of cardiovascular and cerebrovascular diseases. Hydrophilic phenolic acids, including rosmarinic acid(RA) and lithospermic acid B(LAB), are its primary medicinal ingredients. However, the biosynthetic pathway of RA and LAB in S. miltiorrhiza is still poorly understood. In the present study, we accomplished the isolation and characterization of a novel S. miltiorrhiza Hydroxyphenylpyruvate reductase(HPPR) gene, Sm HPPR, which plays an important role in the biosynthesis of RA. Sm HPPR contained a putative catalytic domain and a NAD(P)H-binding motif. The recombinant Sm HPPR enzyme exhibited high HPPR activity, converting 4-hydroxyphenylpyruvic acid(p HPP) to 4-hydroxyphenyllactic acid(p HPL), and exhibited the highest affinity for substrate 4-hydroxyphenylpyruvate. Sm HPPR expression could be induced by various treatments, including SA, GA_3, Me JA and Ag^+, and the changes in Sm HPPR activity were correlated well with hydrophilic phenolic acid accumulation. Sm HPPR was localized in cytoplasm, most likely close to the cytosolic NADPH-dependent hydroxypyruvate reductase active in photorespiration. In addition, the transgenic S. miltiorrhiza hairy roots overexpressing Sm HPPR exhibited up to 10-fold increases in the products of hydrophilic phenolic acid pathway. In conclusion, our findings provide a new insight into the synthesis of active pharmaceutical compounds at molecular level.

Biosynthesis and regulation of diterpenoids in medicinal plants

Plant diterpenoids are widely distributed and abundant natural products with diverse structures and functions in nature,which have been commonly used in pharmaceutical,agricultural and industrial production.In recent years,plant diterpenoids have attracted increasing attention,including their biosynthetic pathways,transcriptional regulatory networks,and biological functions.Herein,the biosynthetic pathways of diterpenoids are summarized in a modular fashion.Further,the regulatory network between diterpene biosynthesis and environmental factors is reviewed.Insights into diterpene metabolism may drive elucidation of complex active diterpene pathways and serve as a knowledge repository for metabolic engineering and cell factory construction.

Phytohormones Jasmonic Acid,Salicylic Acid,Gibberellins,and Abscisic Acid are Key Mediators of Plant Secondary Metabolites

Until recently,many studies on the role of phytohormones in plant secondary metabolism focused on jasmonic acid(JA),salicylic acid(SA),gibberellins(GA),and abscisic acid(ABA).It is now clear that phytohormone?induced regulation of signaling occurs via regulation of the biosynthetic pathway genes at the transcriptional level or through posttranslational regulation,or an increase in secondary metabolite deposition(e.g.,trichomes).Here,we summarize recent advances,updating the current reports on the molecular machinery of phytohormones JA,SA,GA,and ABA involved in plant secondary metabolites.This review emphasizes the differences and similarities among the four phytohormones in regulating various secondary metabolic biosynthetic pathways and also provides suggestions for further research.

Pollen Morphology of Acinos Miller Species Growing in Turkey

The pollen morphology of six taxa of the genus Acinos Miller has been examined under scanning electron microscopy （SEM） and a description of each taxon has been given. Acinos pollen is stephanocolpate （hexacolpate）. Two main exine sculpturing types, foveolate-reticulate （only in subspecies of A. troodi） and reticulate have been defined. The dimension for the polar length ranges between 25.8-47.4 μm, equatorial width 24.5-34.4μm, colpus length 20.0-40.5μm and colpus width 1.2-2.5 μm. The shape is mostly subprolate to prolate, seldomly prolate-spheroidal. The results reveal rather uniform morphological features, however fine details are characteristic to differentiate the pollen taxa. Acinos pollen also share some common morphological features with the other Lamiaceae pollen.

Phytochemical profile and ABTS cation radical scavenging,cupric reducing antioxidant capacity and anticholinesterase activities of endemic Ballota nigra L.subsp.anatolica P.H.Davis from Turkey

Objective:To evaluate the chemical compositions and biological activities of an endemic Ballota nigra L.subsp.anatolica P.H.Davis.Methods:Essential oil and fatty acid composition were determined by GC/MS analysis.ABTS cation radical decolourisation and cupric reducing antioxidant capacity assays were carried out to indicate the antioxidant activity.The anticholinesterase potential of the extracts were determined by Ellman method.Results:The major compounds in the fatty acid composition of the petroleum ether extract were identified as palmitic(36.0%)and linoleic acids(14.3%).The major components of essential oil were 1-hexacosanol(26.7%),germacrene-D(9.3%)and caryophyllene oxide(9.3%).The water extract indicated higher ABTS cation radical scavenging activity than α-tocopherol and BHT,at 100μg/mL.The acetone extract showed 71.58 and 44.71%inhibitory activity against butyrylcholinesterase and acetylcholinesterase enzyme at 200μg/mL,respectively.Conclusions:The water and acetone extracts of Ballota nigra subsp.anatolica can be investigated in terms of both phytochemical and biological aspects to find natural active compounds.

TRICHOMEAND ARTEMISININ REGULATOR 1 Is Required for Trichome Development and Artemisinin Biosynthesis in Artemisia annua

Trichomes, small protrusions on the surface of many plant species, can produce and store various secondary metabolic products. Artemisinin, the most famous and potent medicine for malaria, is synthesized, stored, and secreted by Artemisia annua trichomes. However, the molecular basis regulating the biosynthesis of artemisinin and the development of trichomes in A. annua remains poorly understood. Here, we report that an AP2 transcription factor, TRICHOME AND ARTEMISININ REGULATOR 1 （TAR1）, plays crucial roles in regulating the development of trichomes and the biosynthesis of artemisinin in A. annua. TAR1, which encodes a protein specially located in the nucleus, is mainly expressed in young leaves, flower buds, and some trichomes. In TAR1-RNAi lines, the morphology of trichomes and the composition of cuticular wax were altered, and the artemisinin content was dramatically reduced, which could be significantly increased by TAR1 oeverexpression. Expression levels of several key genes that are involved in artemisinin biosynthesis were altered when TAR1 was silenced or overexpressed. By the electrophoretic mobility shift, yeast one-hybrid and transient transformation β-glucuronidase assays, we showed that ADS and CYP71AV1, two key genes in the biosynthesis pathway of artemisinin, are likely the direct targets of TAR1. Taken together, our results indicate that TAR1 is a key component of the molecular network regulating trichome development and artemisinin biosynthesis in A. annua.

The phenylalanine ammonia-lyase gene family in Isatis indigotica Fort.:molecular cloning,characterization,and expression analysis

Phenolic compounds, metabolites of the phenylpropanoid pathway, play an important role in the growth and environmental adaptation of many plants. Phenylalanine ammonia-lyase(PAL) is the first key enzyme of the phenylpropanoid pathway. The present study was designed to investigate whether there is a multi-gene family in I. Indigotic and, if so, to characterize their properties. We conducted a comprehensive survey on the transcription profiling database by using tBLASTn analysis. Several bioinformatics methods were employed to perform the prediction of composition and physicochemical characters. The expression levels of IiPAL genes in various tissues of I. indigotica with stress treatment were examined by quantitative real-time PCR. Protoplast transient transformation was used to observe the locations of IiPALs. IiPALs were functionally characterized by expression with pET-32a vector in Escherichia colis strain BL21(DE3). Integration of transcripts and metabolite accumulations was used to reveal the relation between IiPALs and target compounds. An new gene(IiPAL2) was identified and both IiPALs had the conserved enzymatic active site Ala-Ser-Gly and were classified as members of dicotyledon. IiPAL1 and IiPAL2 were expressed in roots, stems, leaves, and flowers, with the highest expression levels of IiPAL1 and IiPAL2 being observed in stems and roots, respectively. The two genes responded to the exogenous elicitor in different manners. Subcellular localization experiment showed that both IiPALs were localized in the cytosol. The recombinant proteins were shown to catalyze the conversion of L-Phe to trans-cinnamic acid. Correlation analysis indicated that Ii PAL1 was more close to the biosynthesis of secondary metabolites than IiPAL2. In conclusion, the present study provides a basis for the elucidation of the role of Ii PALs genes in the biosynthesis of phenolic compounds, which will help further metabolic engineering to improve the accumulation of bioactive components in I.indigotica.

IiWRKY34 positively regulates yield, lignan biosynthesis and stress tolerance in Isatis indigotica

Yield potential,pharmaceutical compounds production and stress tolerance capacity are 3 classes of traits that determine the quality of medicinal plants.The autotetraploid Isatis indigotica has greater yield,higher bioactive lignan accumulation and enhanced stress tolerance compared with its diploid progenitor.Here we show that the transcription factor IiWRKY34,with higher expression levels in tetraploid than in diploid I.indigotica,has large pleiotropic effects on an array of traits,including biomass growth rates,lignan biosynthesis,as well as salt and drought stress tolerance.Integrated analysis of transcriptome and metabolome profiling demonstrated that IiWRKY34 expression had far-reaching consequences on both primary and secondary metabolism,reprograming carbon flux towards phenylpropanoids,such as lignans and flavonoids.Transcript-metabolite correlation analysis was applied to construct the regulatory network of IiWRKY34 for lignan biosynthesis.One candidate target Ii4CL3,a key rate-limiting enzyme of lignan biosynthesis as indicated in our previous study,has been demonstrated to indeed be activated by IiWRKY34.Collectively,the results indicate that the differentially expressed IiWRKY34 has contributed significantly to the polyploidy vigor of I.indigotica,and manipulation of this gene will facilitate comprehensive improvements of I.indigotica herb.

The transcription factors TLR1 and TLR2 negatively regulate trichome density and artemisinin levels in Artemisia annua

The important antimalarial drug artemisinin is biosynthesized and stored in Artemisia annua glandular trichomes and the artemisinin content correlates with trichome density;however,the factors affecting trichome development are largely unknown.Here,we demonstrate that the A.annua R2R3 MYB transcription factor TrichomeLess Regulator 1(TLR1)negatively regulates trichome development.In A.annua,TLR1 overexpression lines had 44.7%–64.0%lower trichome density and 11.5%–49.4%lower artemisinin contents and TLR1-RNAi lines had 33%–93.3%higher trichome density and 32.2%–84.0%higher artemisinin contents compared with non-transgenic controls.TLR1 also negatively regulates the expression of anthocyanin biosynthetic pathway genes in A.annua.When heterologously expressed in Arabidopsis thaliana,TLR1 interacts with GLABROUS3a,positive regulator of trichome development,and represses trichome development.Yeast two-hybrid and pull-down assays indicated that TLR1 interacts with the WUSCHEL homeobox(WOX)protein AaWOX1,which interacts with the LEAFY-like transcription factor TLR2.TLR2 overexpression in Arabidopsis and A.annua showed that TLR2 reduces trichome development by reducing gibberellin levels.Furthermore,artemisinin contents were 19%–43%lower in TLR2-overexpressing A.annua plants compared to controls.These data indicate that TLR1 and TLR2 negatively regulate trichome density by lowering gibberellin levels and may enable approaches to enhance artemisinin yields.