Jasmonic acid regulates the biosynthesis of medicinal metabolites via the JAZ9-MYB76 complex in Salvia miltiorrhiza

Jasmonic acid(JA)signaling pathway plays an important role in tanshinone and phenolic acid biosynthesis in Salvia miltiorrhiza.However,the specific regulatory mechanism remains largely unclear.Previous work showed that a JASMONATE ZIM-domain(JAZ)protein,SmJAZ9,acted as a repressor of tanshinone production in S.miltiorrhiza.In this study,we revealed that SmJAZ9 reduced both phenolic acid accumulation and related biosynthetic gene expression,confirming that SmJAZ9 also negatively affected phenolic acid biosynthesis.Then,we identified a novel MYB transcription factor,SmMYB76,which interacted with SmJAZ9.SmMYB76 repressed phenolic acid biosynthesis by directly downregulating SmPAL1,Sm4CL2,and SmRAS1.Further investigation demonstrated that JA mediated phenolic acids biosynthesis via SmJAZ9-SmMYB76 complex.Taken together,these findings state the molecular mechanism that SmJAZ9-SmMYB76 regulated phenolic acid biosynthesis at the transcriptional and protein levels,which provided new insights into JA signaling pathway regulating plant metabolism.

Larvicidal activities of hydro-ethanolic extracts of three Cameroonian medicinal plants against Aedes albopictus

Objective: To investigate the larvicidal activity of Syzygium guineense(Myrtaceae)(S. guineense), Monodora myristica and Zanthoxylum heitzii(Rutaceae)(Z. heitzii) extracts against Aedes albopictus(Ae. albopictus).Methods: The larvicidal activity of the hydro-ethanolic extracts from these plant species was assessed at three different concentrations(50, 100 and 200 mg/L) on first-instar of Ae. albopictus larvae in comparison with untreated controls. Mortality rate was recorded daily for a period of 12 days. The values of LC_(50) and lethal time killing 50% of the tested individuals(LT_(50)) were calculated using the log-probit analysis.Results: The root extract of S. guineense exhibited the best activity with 100% mortality after 8 days of treatment at 200 mg/L, followed by the fruit extract of Z. heitzii with83.33% mortality at the same concentration. Nonetheless, larvae were most susceptible to the fruit extract of Z. heitzii both in terms of LC_(50)(39.89 mg/L) and LT_(50)(145.68 h). A statistically significant difference between the control and the group treated at 200 mg/L was noticed in all the extracts.Conclusions: The present study shows that the hydro-ethanolic extracts of S. guineense,Monodora myristica and Z. heitzii tested have significant larvicidal activity. These preliminary results are of great interest and some of these plant species can be proposed for the formulation of new bioinsecticides to control Ae. albopictus populations.

Multilayered regulation of secondary metabolism in medicinal plants

Medicinal plants represent a huge reservoir of secondary metabolites(SMs),substances with significant pharmaceutical and industrial potential.However,obtaining secondary metabolites remains a challenge due to their low-yield accumulation in medicinal plants;moreover,these secondary metabolites are produced through tightly coordinated pathways involving many spatiotemporally and environmentally regulated steps.The first regulatory layer involves a complex network of transcription factors;a second,more recently discovered layer of complexity in the regulation of SMs is epigenetic modification,such as DNA methylation,histone modification and small RNA-based mechanisms,which can jointly or separately influence secondary metabolites by regulating gene expression.Here,we summarize the findings in the fields of genetic and epigenetic regulation with a special emphasis on SMs in medicinal plants,providing a new perspective on the multiple layers of regulation of gene expression.

The transcription factor OpWRKY2 positively regulates the biosynthesis of the anticancer drug camptothecin in Ophiorrhiza pumila

The limited bioavailability of plant-derived natural products with anticancer activity poses major challenges to the pharmaceutical industry.An example of this is camptothecin,a monoterpene indole alkaloid with potent anticancer activity that is extracted at very low concentrations from woody plants.Recently,camptothecin biosynthesis has been shown to become biotechnologically amenable in hairy-root systems of the natural producer Ophiorrhiza pumila.Here,time-course expression and metabolite analyses were performed to identify novel transcriptional regulators of camptothecin biosynthesis in O.pumila.It is shown here that camptothecin production increased over cultivation time and that the expression pattern of the WRKY transcription factor encoding gene OpWRKY2 is closely correlated with camptothecin accumulation.Overexpression of OpWRKY2 led to a more than three-fold increase in camptothecin levels.Accordingly,silencing of OpWRKY2 correlated with decreased camptothecin levels in the plant.Further detailed molecular characterization by electrophoretic mobility shift,yeast one-hybrid and dual-luciferase assays showed that OpWRKY2 directly binds and activates the central camptothecin pathway gene OpTDC.Taken together,the results of this study demonstrate that OpWRKY2 acts as a direct positive regulator of camptothecin biosynthesis.As such,a feasible strategy for the over-accumulation of camptothecin in a biotechnologically amenable system is presented.

Three new C_(21) steroidal glycosides from the roots of Cynanchum inamoenum

Three new C21 steroidal glycosides named inamoside E （1）, inamoside F （2） and inamoside G （3） were isolated from the roots of Cynanchum inamoenum （Maxim.） Loes. Their structures were determined by spectroscopic analysis, especially by ID and 2D NMR experiments.

AaWRKY17, a positive regulator of artemisinin biosynthesis, is involved in resistance to Pseudomonas syringae in Artemisia annua

Artemisia annua,a traditional Chinese medicinal plant,remains the only plant source for artemisinin production,yet few genes have been identified to be involved in both the response to biotic stresses,such as pathogens,and artemisinin biosynthesis.Here,we isolated and identified the WRKY transcription factor(TF)AaWRKY17,which could significantly increase the artemisinin content and resistance to Pseudomonas syringae in A.annua.Yeast one-hybrid(Y1H),dual-luciferase(dual-LUC),and electrophoretic mobility shift assay(EMSA)results showed that AaWRKY17 directly bound to the W-box motifs in the promoter region of the artemisinin biosynthetic pathway gene amorpha-4,11-diene synthase(ADS)and promoted its expression.Real-time quantitative PCR(RT-qPCR)analysis revealed that the transcript levels of two defense marker genes,Pathogenesis-Related 5(PR5)and NDR1/HIN1-LIKE 10(NHL10),were greatly increased in AaWRKY17-overexpressing transgenic A.annua plants.Additionally,overexpression of AaWRKY17 in A.annua resulted in decreased susceptibility to P.syringae.These results indicated that AaWRKY17 acted as a positive regulator in response to P.syringae infection.Together,our findings demonstrated that the novel WRKY transcription factor AaWRKY17 could potentially be used in transgenic breeding to improve the content of artemisinin and pathogen tolerance in A.annua.

Effects of Drought Stress on Morphological and Physiological Indices of New Coix Varieties in Yunnan

In order to study the effects of drought stress on the morphological and physiological indexes of Yunnan Coix at different growth stages, two new varieties of Yunnan Coix Wenyi 4 and Wenyi 5 were used as experimental materials, potted and compared with CK by normal irrigation. Morphological indices such as plant height, stem diameter and physiological indices such as protein, soluble sugar, chlorophyll content and relative conductivity (REC) of the two varieties under drought stress at seedling stage, tillering stage and jointing stage were determined, and data statistics and variance analysis were performed for each index. The results showed that the morphological indices, plant height of Wenyi 4 and Wenyi 5 were significantly affected by drought stress at seedling stage, while the stem diameter was significantly affected by drought stress at seedling and jointing stage. In terms of physiological indices, the relative conductivity, protein and soluble sugar contents of Wenyi 5 increased at a higher degree, whereas chlorophyll content decreased at a higher degree, indicating that Wenyi 5 was greatly affected by drought stress. Through the analysis of drought resistance of the tested materials at different stages by polar ordination method, the results were as follows: jointing stage of Wenyi 5>jointing stage of Wenyi 4>seedling stage of Wenyi 4>seedling stage of Wenyi 5>tillering Stage of Wenyi 4>Tillering Stage of Wenyi 5. For the growth period, the drought resistance was as follows: jointing stage>seedling stage>tillering stage. In conclusion, Wenyi 4 has strong drought resistance and is suitable for droughtresistant cultivation.

Study on the Effects of Reducing Blood Lipid by Flavonoids from Ampelopsis grossedentata on Hyperlipidemia Rats

In order to study the effects of Ampelopsis grossedentata on endogenous cholesterol biosynthesis and the effects of reducing blood lipid in hyperlipidemia rats,extraction of flavonoids from Ampelopsis grossedentata by using ethanol(TFAG),and the effects of TFAG on intracellular cholesterol synthesis were detected by amphotericin B-cell model;SD hyperlipidemia rat model was established by feeding high fat diet.A formulated medicine called Xuezhikang was used as a positive control,and TFAG of different doses were administered to the stomach for 30 d continuously to measure the indexes of heart,liver tissue homogenate and serum;part of the liver was taken for pathological observation.The results showed that TFAG could significantly inhibit the synthesis of cholesterol in cells.TFAG of different doses could significantly reduce the content of TC and TG in serum of hyperlipidemia rats,and increase the content of HDL;TC and TG in heart and liver were also decreased;besides,it could increase the content of SOD,CAT and GSH in the liver of hyperlipidemia rats,and reduce the content of MDA.The results of pathological section showed that TFAG could improve the damage degree of hepatocytes in hyperlipidemia rats,and the effect of high dose group was similar to that of Xuezhikang group.In general,TFAG has good antioxidant and reducing blood lipid effects,and can effectively inhibit liver steatosis.

Solvent-free synthesis of some ethyl arylglyoxylates

An efficient and convenient approach to synthesize of ethyl arylglyoxylates by solvent-free grinding was described. Eight ethyl arylglyoxylates had been synthesized by this method, this method provided several advantages such as little pollution, high yield, good selectivity, and simple workup procedure, their structures were confirmed by IR, ^1HNMR.

Increased artemisinin production by promoting glandular secretory trichome formation and reconstructing the artemisinin biosynthetic pathway in Artemisia annua

Dear Editor,Artemisinin,which has potent antimalarial properties,is a sesquiterpene endoperoxide originally isolated from the traditional Chinese medicinal plant Artemisia annua.However,the artemisinin content in wild-type(WT)A.annua is low(1-10 mg/g dry weight),leading to its erratic supply and price fluctuations[1].

Biosynthetic pathway of prescription cucurbitacin IIa and high-level production of key triterpenoid intermediates in engineered yeast and tobacco

Cucurbitacin IIa is a triterpenoid isolated exclusively from Hemsleya plants and a non-steroidal anti-inflammatory drug that functions as the main ingredient of prescription Hemslecin capsules and tablets in China.Synthetic biology provides new strategies for production of such valuable cucurbitacins at a large scale;however,the biosynthetic pathway of cucurbitacin IIa has been unknown,and the heterologous production of cucurbitacins in galactose medium has been expensive and low yielding.In this study,we characterized the functions of genes encoding two squalene epoxidases(HcSE1-2),six oxidosqualene cyclases(HcOSC1-6),two CYP450s(HcCYP87D20 and HcCYP81Q59),and an acyltransferase(HcAT1)in cucurbitacin IIa biosynthesis by heterologous expression in Saccharomyces cerevisiae and Nicotiana benthamiana.We achieved high-level production of the key cucurbitacin precursor 11-carbonyl-20b-hydroxy-Cuol from glucose in yeast via modular engineering of the mevalonate pathway and optimization of P450 expression levels.The resulting yields of 46.41 mg/l 11-carbonyl-20b-hydroxy-Cuol and 126.47 mg/l total cucurbitacin triterpenoids in shake flasks are the highest yields yet reported from engineered microbes.Subsequently,production of 11-carbonyl-20b-hydroxy-Cuol by transient gene expression in tobacco resulted in yields of 1.28 mg/g dry weight in leaves.This work reveals the key genes involved in biosynthesis of prescription cucurbitacin IIa and demonstrates that engineered yeast cultivated with glucose can produce high yields of key triterpenoid intermediates.We describe a low-cost and highly efficient platform for rapid screening of candidate genes and high-yield production of pharmacological triterpenoids.

The methyl jasmonate-responsive transcription factor SmMYB1 promotes phenolic acid biosynthesis in Salvia miltiorrhiza

Water-soluble phenolic acids are major bioactive compounds in the medicinal plant species Salvia miltiorrhiza.Phenolic acid biosynthesis is induced by methyl jasmonate(MeJA)in this important Chinese herb.Here,we investigated the mechanism underlying this induction by analyzing a transcriptome library of S.miltiorrhiza in response to MeJA.Global transcriptome analysis identified the MeJA-responsive R2R3-MYB transcription factor-encoding gene SmMYB1.Overexpressing SmMYB1 significantly promoted phenolic acid accumulation and upregulated the expression of genes encoding key enzymes in the phenolic acid biosynthesis pathway,including cytochrome P450-dependent monooxygenase(CYP98A14).Dual-luciferase(dual-LUC)assays and/or an electrophoretic mobility shift assays(EMSAs)indicated that SmMYB1 activated the expression of CYP98A14,as well as the expression of genes encoding anthocyanin biosynthesis pathway enzymes,including chalcone isomerase(CHI)and anthocyanidin synthase(ANS).In addition,SmMYB1 was shown to interact with SmMYC2 to additively promote CYP98A14 expression compared to the action of SmMYB1 alone.Taken together,these results demonstrate that SmMYB1 is an activator that improves the accumulation of phenolic acids and anthocyanins in S.miltiorrhiza.These findings lay the foundation for in-depth studies of the molecular mechanism underlying MeJA-mediated phenolic acid biosynthesis and for the metabolic engineering of bioactive ingredients in S.miltiorrhiza.

Nanotechnologies in Food Science:Applications,Recent Trends,and Future Perspectives

Nanotechnology is a key advanced technology enabling contribution,development,and sustainable impact on food,medicine,and agriculture sectors.Nanomaterials have potential to lead qualitative and quantitative production of healthier,safer,and high-quality functional foods which are perishable or semi-perishable in nature.Nanotechnologies are superior than conventional food processing technologies with increased shelf life of food products,preventing contamination,and production of enhanced food quality.This comprehensive review on nanotechnologies for functional food development describes the current trends and future perspectives of advanced nanomaterials in food sector considering processing,packaging,security,and storage.Applications of nanotechnologies enhance the food bioavailability,taste,texture,and consistency,achieved through modification of particle size,possible cluster formation,and surface charge of food nanomaterials.In addition,the nanodelivery-mediated nutraceuticals,synergistic action of nanomaterials in food protection,and the application of nanosensors in smart food packaging for monitoring the quality of the stored foods and the common methods employed for assessing the impact of nanomaterials in biological systems are also discussed.

OpNAC1 transcription factor regulates the biosynthesis of the anticancer drug camptothecin by targeting loganic acid O-methyltransferase in Ophiorrhiza pumila

Camptothecin(CPT) is an anticancer pentacyclic quinoline alkaloid widely used to treat cancer patients worldwide. However, the biosynthetic pathway and transcriptional regulation of camptothecin are largely unknown. Ophiorrhiza pumila, the herbaceous plant from the Rubiaceae family, has emerged as a model plant for studying camptothecin biosynthesis and regulation. In this study, a high-quality reference genome of O. pumila with estimated size of ~456.90Mb was reported, and the accumulation level of camptothecin in roots was higher than that in stems and leaves. Based on its spatial distribution in the plant, we examined gene functions and expression by combining genomics with transcriptomic analysis.Two loganic acid O-methyltransferase(OpLAMTs)were identified in strictosidine-producing plant O.pumila, and enzyme catalysis assays showed that OpLAMT1 and not OpLAMT2 could convert loganic acid into loganin. Further knock-out of OpL AMT1expression led to the elimination of loganin and camptothecin accumulation in O. pumila hairy roots.Four key residues were identified in OpLAMT1 protein crucial for the catalytic activity of loganic acid to loganin. By co-expression network, we identified a NAC transcription factor, OpNAC1, as a candidate gene for regulating camptothecin biosynthesis.Transgenic hairy roots and biochemical assays demonstrated that OpNAC1 suppressed OpLAMT1 expression. Here, we reported on two camptothecin metabolic engineering strategies paving the road for industrial-scale production of camptothecin in CPT-producing plants.

Comparative genomics reveals the diversification of triterpenoid biosynthesis and origin of ocotilloltype triterpenes in Panax

Gene duplication is assumed to be the major force driving the evolution of metabolite biosynthesis in plants.Freed from functional burdens,duplicated genes can mutate toward novelties until fixed due to selective fitness.However,the extent to which this mechanism has driven the diversification of metabolite biosynthesis remains to be tested.Here we performed comparative genomics analysis and functional characterization to evaluate the impact of gene duplication on the evolution of triterpenoid biosynthesis using Panax species as models.Wefound that whole-genome duplications(WGDs)occurred independently in Araliaceae and Apiaceae lineages.Comparative genomics revealed the evolutionary trajectories of triterpenoid biosynthesis in plants,which was mainly promoted by WGDs and tandem duplication.Lanosterol synthase(LAS)was likely derived from a tandemduplicate of cycloartenol synthase that predated the emergence of Nymphaeales.Under episodic diversifying selection,the LAS gene duplicates produced by g whole-genome triplication have given rise to triterpene biosynthesis in core eudicots through neofunctionalization.Moreover,functional characterization revealed that oxidosqualene cyclases(OSCs)responsible for synthesizing dammarane-type triterpenes in Panax species were also capable of producing ocotilloltype triterpenes.Genomic and biochemical evidence suggested that Panax genes encoding the above OSCs originated from the specialization of one OSC gene duplicate produced from a recent WGD shared by Araliaceae(Pg-b).Our results reveal the crucial role of gene duplication in diversification of triterpenoid biosynthesis in plants and provide insight into the origin of ocotillol-type triterpenes in Panax species.

Constructing cactus-like mixed dimensional MOF@MOF as sorbent for extraction of bisphenols from environmental water

Metal-organic frameworks(MOFs)received considerable attention to adsorption and removal of various environmental pollutants because of some inherent advantages.However,it is challenging but meaningful to design and fabricate hierarchical mixed-dimensional MOFs with synergistic effects to enhance the performance for removal and preconcentration of environmental pollutants.Herein,a new hierarchical two-dimensional(2D)-three-dimensional(3D)mixed-dimensional cactus-like MOF@MOF hybrid material(PCN-134@Zr-BTB)was prepared by in-situ growth of 2D MOF nanosheets(Zr-BTB)on the surface of 3D MOF(PCN-134).The PCN-134@Zr-BTB composites combine the advantages of 2D and 3D MOFs with extensive mesoporous structures and large surface area for effective removal and enrichment of bisphenols(BPs).In comparison with pristine PCN-134 and Zr-BTB materials,the PCN-134@Zr-BTB hybrid material presented excellent adsorption performance for BPs.The adsorption isotherms are consistent with the Langmuir model,and the maximum adsorption capacity of four bisphenols(BPs)ranged from 135.1 mg/g to 628.9 mg/g.The adsorption kinetics are in accordance with the pseudo-second-order model.The recoveries ranged from 72.8%to 108%.The limits of detection were calculated at 0.02-0.03 ng/mL.The enrichment factors were calculated in the range of 310-374.According to FT-IR and XPS analysis,the main adsorption mechanisms are hydrogen bonding and π-π stacking.Nevertheless,this work provides a new and convenient strategy for the preparation of new hierarchical mixed-dimensional MOF@MOF(PCN-134@Zr-BTB)hybrid material for extraction and enrichment of BPs from aqueous matrix.

Water–soluble and polarity–sensitive near–infrared fluorescent probe for long–time specific cancer cell membranes imaging and C. Elegans label

A novel D–π –A structure and near–infrared fluorescent probe(DCITT) with high polarity sensitivity and membrane targeting was reported. The fluorescent spectra of DCITT were polarity dependent and Stokes shift was greater than 300 nm. Due to its high fluorescence quantum yield, low cytotoxicity and photostability, DCITT could be used as a labeling probe in multicellular organisms. In particular, DCITT effectively distinguished tumor cells from normal cells because it could specifically light up the cancer cells membrane based on strong red fluorescence for a long time. On this basis, a polar–sensitive cell membrane probe is developed to differentiate tumor cells from normal cells, which provides an idea and method for the early diagnosis of tumor at cellular level.

The only purple-flower species in Actaea L.is pollinated by a hornet

Examining the pollination biology of plant species is not only crucial for enhancing our understanding of their reproductive biology,but also essential for elucidating their adaptation and evolutionary history.Here,we investigated the breeding system and pollination biology of two closely related species in Actaea.The flower of A.purpurea is unique in the genus with purple and chartaceous(paper-like)sepals,fewer stamens with yellow anthers and purple filaments.Through three seasons of field observation and exclusion experiments,we determined that A.purpurea was primarily pollinated by a hornet species,Vespa bicolor,which also served as the most efficient pollinator.In contrast,A.japonica was primarily pollinated by large flies.Actaea purpurea exhibited a significantly higher cumulative nectar volume than A.japonica,which could be a crucial factor in attracting V.bicolor.A control experiment further demonstrated that removing the nectar leaf(petal)significantly decreased the visiting frequency of V.bicolor.Breeding system studies revealed that both species were self-compatible,yet they primarily underwent outcrossing in natural habitats.Our study presents a compelling case of possible pollinator shift in A.purpurea accompanied by morphological divergence.A more in-depth investigation of this system would offer crucial insights into the extent to which pollinators are involved in the plant speciation process and whether they contribute to reproductive isolation between closely related species.

SmMYB2 promotes salvianolic acid biosynthesis in the medicinal herb Salvia miltiorrhiza

MYB transcription factors play vital roles in plant growth and metabolism.The phytohormone methyl jasmonate(MeJA)promotes phenolic acid accumulation in the medicinal herb Salvia miltiorrhiza,but the regulatory mechanism is poorly understood.Here,we identified the MeJA-responsive R2R3-MYB transcription factor gene 5mMYB2 from a transcriptome library produced from MeJAtreated S.mi'/horr/ifza hairy roots.Sm/VIYB2 expression was tightly correlated with the expression of key salvianolic acid biosynthetic genes including CYP^8Ai^.5mMYB2 was highly expressed in the periderm of S.miltiorrhiza and SmMYB2 localized to the nucleus.Overexpressing Sml\AYB2 in S.miltiorrhiza hairy roots significantly increased the levels of salvianolic acids(including rosmarinic acid and salvianolic acid B)by upregulating salvianolic acid biosynthetic genes such as CYP98A14.SmMYB2 binds to the MYB-binding motifs in the promoter of CYP98A14,as confirmed by a dual-luciferase assay and electrophoretic mobility shift assays.Anthocyanin contents were significantly higher in Sm/ViyB2-overexpressing hairy root lines than the control,primarily due to the increased expression of CHI,DFR,and ANS.These findings reveal the novel regulatory role of SmMYB2 in MeJA-mediated phenolic acid biosynthesis,providing a useful target gene for metabolic engineering and shedding light on the salvianolic acid regulatory network.

Morphogenesis of Pistillate Flowers of Cercidiphyllum japonicum(Cercidiphyllaceae)

Floral morphogenesis and the development of Cercidiphyllumjaponicum Sieb. et Zucc. were observed by scanning electron microscopy （SEM）. The results showed that the pistillate inflorescences were congested spikes with the flowers arranged opposite. Great differences between the so-called ＂bract＂ and the vegetative leaf were observed both in morphogenesis and morphology. In morphogenesis, the ＂bract＂ primordium is crescent-shaped, truncated at the apex and not conduplicate, has no stipule primordium at the base but does have some inconspicuous teeth in the margin that are not glandular. The leaf primordium is triangular, cycloidal at the apex, conduplicate, has two stipule primordia at the base, has one gland-tooth at the apex occurring at first and some gland-teeth in the margin that occur later. In morphology, the ＂bract＂ is also different to the vegetative leaf in some characteristics that were also illustrated in the present paper. Based on the hypothesis that the bract is more similar to the vegetative leaf than the tepal, we considered that the so-called ＂bract＂ of C.japonicum might be the tepal of the pistillate flower in morphological nature. Therefore, each pistillate flower contains a tepal and a carpel. We did not find any trace of other floral organs in the morphogenesis of the pistillate flower. Therefore we considered that the unicarpellate status of extant Cercidiphyllum might be to highly reduce and advance characteristics that make the extant Cercidiphyllum isolated from both fossil Cercidiphyllum-like plants and its extant affinities.