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Introduction Transcription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants. Objectives We selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering. Methods G-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual-luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism. Results PAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P + T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P + T and PAP1 genotypes. Conclusion G-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.

Background Chronic subdural hematomas (CSDHs) are one of the most common neurosurgical conditions. The standard surgical technique includes burr-hole craniostomy, followed by intraoperative irrigation and placement of subdural closed-system drainage. The drainage is generally removed after 48 hours, which can be described as fixed-time drainage strategy. According to literature, the recurrence rate is 5–33% with this strategy. In our retrospective study, postoperative hematoma volume was found to significantly increase the risk of recurrence. Based on these results, an exhaustive drainage strategy is conducted to minimize postoperative hematoma volume and achieve a low recurrence rate and good outcomes. Methods This is a prospective, multicenter, randomized controlled trial designed to include 304 participants over the age of 18–90 years presenting with a symptomatic CSDH verified on cranial computed tomography or magnetic resonance imaging. Participants will be randomly allocated to perform exhaustive drainage (treatment group) or fixed-time drainage (control group) after one-burr hole craniostomy. The primary endpoint will be recurrence indicating a reoperation within 6 months. Discussion This study will validate the effect and safety of exhaustive drainage after one-burr hole craniostomy in reducing recurrence rates and provide critical information to improve CSDH surgical management. Trial registration Clinicaltrials.gov, NCT04573387. Registered on October 5, 2020. (https://www.clinicaltrials.gov/ct2/show/study/NCT04573387.)

Yiqi Shengsui formula (YQSSF) is a commonly used formula to treat chemotherapy-induced myelosuppression, but little is known about its therapeutic mechanisms.This study aims to examine the effect of YQSSF in treating myelosuppression and explore its mechanism.A myelosuppression BALB/c mouse model was established by intraperitoneal (i.p.) injection of cyclophosphamide (CTX). The efficacy of YQSSF in alleviating chemotherapy-induced myelosuppression was evaluated by blood cell count, immune organ (thymus, spleen, liver) index, bone marrow nucleated cell (BMNC) count and histopathological analysis of bone marrow and spleen. Then, ultra-performance liquid chromatograph quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was performed to analyze the ingredients of YQSSF extract. Key effects and potential mechanism of YQSSF extract in alleviating myelosuppression were predicted by network pharmacology method. Finally, cell cycle and TUNEL staining of bone marrow cells was detected to verify the key effects, and RT-qPCR or Western blotting were performed to measure the gene and protein expressions of the effect targets respectively to confirm the predicted mechanism of YQSSF for myelosuppression.YQSSF up-regulated the number of peripheral blood leukocytes and BMNC, reduced spleen index and liver index, improved the pathological morphology of bone marrow and spleen. A total of 40 ingredients were isolated from YQSSF extract using UPLC-Q/TOF-MS analysis. Network pharmacology revealed that YQSSF regulated both proliferation and apoptosis to alleviate myelosuppression. Finally, YQSSF decreased G0/G1 ratio, increased the proportion of bone marrow cells in S phase and proliferation index (PI), and reduced apoptotic cells in femur bone marrow. RT-qPCR and Western blotting showed that YQSSF up-regulated the expression levels of CDK4, CDK6, CyclinB1, c-Myc and Bcl-2, as well as down-regulated the expression levels of Cyt-c, Fas, Caspase-8/3 and p53.YQSSF promotes the proliferation and inhibits the apoptosis of bone marrow cells to relieve chemotherapy-induced myelosuppression.

The discovery of three iridoid synthases (GjISY, GjISY2 and GjISY4) from Gardenia jasminoides and their functional characterization increase the understanding of iridoid scaffold/iridoid glycoside biosynthesis in iridoid-producing plants. Iridoids are a class of noncanonical monoterpenes that are found naturally in the plant kingdom mostly as glycosides. Over 40 iridoid glycosides (e.g., geniposide, gardenoside and shanzhiside) have been isolated from Gardenia jasminoides. They have multiple pharmacological properties and health-promoting effects. However, their biosynthetic pathway is poorly understood, and the iridoid synthase (ISY) responsible for the cyclization of the core scaffold remains unclear. In this study, three homologs of ISYs from G. jasminoides (GjISY, GjISY2 and GjISY4) were identified on the basis of transcriptomic data and functionally characterized. The genomic structure and intron-exon arrangement revealed that all three ISYs contained an intron. Biochemical assays indicated that all three recombinant enzymes reduced 8-oxogeranial to nepetalactol and its open forms (iridodials) as the products of the classical CrISY (Catharanthus roseus). In addition, all three enzymes reduced progesterone to 5-β-prognane-3,20-dione. However, only GjISY2 and GjISY4 reduced 2-cyclohexen-1-one to cyclohexanone. Overall, the GjISY2 expression levels in the flowers and fruits were similar to the GjISY and GjISY4 expression levels. By contrast, the GjISY2 expression levels in the upper and lower leaves were substantially higher than the GjISY and GjISY4 expression levels. Among the three, GjISY2 exhibited the highest catalytic efficiency for 8-oxogeranial. GjISY2 might be the major contributor to iridoid biosynthesis in G. jasminoides. Collectively, our results advance the understanding of iridoid scaffold/iridoid glycoside biosynthesis in G. jasminoides and provide a potential target for metabolic engineering and breeding.

Chrysanthemum indicum L. is a type of herb that is widely used in China, Korea, and Japan. It has been used as an ingredient in traditional medicines, tea, and functional food because of its various anti-inflammatory and anti-oxidant bioactivities. Such bioactivities have been associated with flavonoids such as apigenin, luteolin and linarin in C. indicum. However, the biosynthesis pathway has not been investigated. In this study, using transcriptomic analysis and targeted metabolic profiling from five different tissues, we characterize the levels of flavonoids and mine the corresponding genes involved in flavonoid biosynthesis. Transcriptomic analysis revealed that 103 unigenes are involved in flavonoid-related biosynthesis pathways. Flavone synthase (FNS) is the key enzyme responsible for flavone synthesis and provides precursors for acacetin and linarin biosynthesis. One putative FNS Ⅱ gene, with the highest Reads Per Kilobase per Million mapped reads (RPKM) in flower and flower bud was cloned. Quantitative real-time polymerase chain reaction (RT-qPCR) revealed that CiFNSⅡ exhibited a similar expression pattern to that in the transcriptome in terms of RPKM. In addition, a targeted metabolic profiling of three flavanones (naringenin, eriodictyol, and liquiritigenin), three flavones (apigenin, luteolin, and 7,4′-dihydroxyflavone), and two flavone derivatives (linarin and acacetin) was performed to characterize the distribution of these flavonoids in different tissues of C. indicum. The recombinant FNSⅡ protein expressed in yeast was able to catalyze the conversion of three flavanones into the respective flavones. Based on the transcriptome analysis, metabolic profiling, and activity assays, a linarin biosynthesis pathway is proposed. Our study provides insight into the potential application of molecular breeding and metabolic engineering for improving the quality of cultivated C. indicum.

Iridoid compounds have been reported to be accumulated in the dried fruits of Gardenia jasminoides (Rubiaceae), which are used in traditional Chinese medicine. These compounds exhibit obvious pharmacological activities, including effective protective effects on the liver and therapeutic efficacy for cardiovascular diseases. However, the biosynthetic pathway of iridoid remains uninvestigated. In this study, 12 transcriptomes from four tissues (lower leaves, top leaves, flowers, and fruits) were analyzed to characterize the accumulation of active constituents and to identify the corresponding genes involved in iridoid biosynthesis. Almost all genes in the mevalonate (MVA) pathway and the 2-C-methyl- d -erythritol-4-phosphate (MEP) pathway were found in our transcriptome database; these pathways comprise upstream pathways of iridoid compounds (monoterpene derivatives), most of which are abundant in flowers and fruits. Geraniol synthase (GES), which is involved in the first branch step in the iridoid biosynthetic pathway, was cloned, and quantitative real-time PCR (qRT-PCR) revealed that GjGES showed a similar expression pattern to that in the transcriptome in terms of reads per kilobase per million (RPKM) mapped reads. The recombinant GjGES protein efficiently converted geranyl diphosphate (GPP) to geraniol. Subsequently, the metabolic profiling of seven iridoids and iridoid glycosides, namely geniposide, gardenoside, genipin, geniposidic acid, genipin-1-β-gentiobioside, shanzhiside, and shanzhiside methyl ester, revealed that these compounds were highly accumulated in fruits. Consequently, the metabolites and speculated unigenes involved in iridoid metabolic pathways enabled the identification of glucosyltransferase, hydroxylase, and O-methyltransferase candidate genes responsible for the biosynthesis of iridoid and iridoid glycosides. The results of the present study can serve as a reference for the functional characterization of enzyme-coding genes and are beneficial for the engineering of biosynthetic pathways of iridoids and iridoid glycosides in the future.

The fruits of Amomum villosum and Amomum longiligulare are both used medicinally as Fructus Amomi the famous traditional Chinese medicine, however, the medicinal quality of A. villosum is better than that of A. longiligulare. Volatile terpenoids in the seeds, especially bornyl acetate and borneol, are the medicinal components of Fructus Amomi. The volatile terpenoids and transcriptome of developing seeds of A. villosum and A. longiligulare were compared in this study. The result revealed that the bornyl acetate and borneol contents were higher in A. villosum than in A. longiligulare. Additionally, six terpenoid synthase genes (AlTPS1–AlTPS6) were screened from the transcriptome of A. longiligulare, and AlTPS2 and AlTPS3 were found to share 98 and 83% identity with AvTPS2 and AvBPPS (bornyl diphosphate synthase) from A. villosum, respectively. BPPS is the key enzyme for the biosynthesis of borneol and bornyl acetate. Biochemical assays improved that AlTPS2 had an identical function to AvTPS2 as linalool synthase; however, AlTPS3 produced camphene as the major product and bornyl diphosphate (BPP) as the secondary product, whereas AvBPPS produced BPP as its major product. There was only one different amino acid between AlTPS3 (A496) and AvBPPS (G495) in their conserved motifs, and the site-directed mutation of A496G in DTE motif of AlTPS3 changed the major product from camphene to BPP. Molecular docking suggests that A496G mutation narrows the camphene-binding pocket and decreases the BPP-binding energy, thus increases the product BPP selectivity of enzyme. In addition, the expression level of AvBPPS was significantly higher than that of AlTPS3 in seeds, which was consistent with the related-metabolites contents. This study provides insight into the TPS-related molecular bases for the biosynthesis and accumulation differences of the bioactive terpenoids between A. villosum and A. longiligulare. BPPS, the key gene involved in the biosynthesis of the active compound, was identified as a target gene that could be applied for the quality-related identification and breeding of Fructus Amomi.

Transcription factors (TFs) andWe selected two Arabidopsis TF types and two tobacco CRE types to design aG-box and MYB recognition elements (MREs) were identified in fourPAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activatedG-boxes, MREs, and two TF types are appropriate molecular tools for a

IntroductionTranscription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants.ObjectivesWe selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering.MethodsG-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism.ResultsPAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P+T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P+T and PAP1 genotypes.ConclusionG-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.

Chrysanthemum indicum has long been used in traditional Chinese medicine for its health-promoting benefits. Studies on C. indicum have mainly focused on the flowers. Terpenoid distribution in various parts of the plant and characterization of terpene synthases remain unclear. In this study, volatile metabolic profiling was performed to compare the composition and quantity of terpenoids distributed in the root, stem, leaf, flower bud and flower of C. indicum. The potential for extracting active ingredients from the root, stem, and leaf was also examined. In total, 17 monoterpenoids and 27 sesquiterpenoids were identified. Transcriptome data were used to clone two monoterpene synthases and two sesquiterpene synthases highly expressed in the root. The recombinant proteins of full-length and truncated versions of C. indicum terpene synthase (CiTPS1) produced α-pinene, but the truncated one was catalytically more efficient than the full-length version. No product could be detected when full-length version of CiTPS2 was used for catalyzing GPP, but the truncated one can produce a minor amount of α-pinene. CiTPS3 contributed to the production of three sesquiterpenoids, namely β-farnesene, petasitene, and α-bisabolene. CiTPS4 acted as a difunctional enzyme, contributing to the production of four monoterpenoids and three sesquiterpenoids, including petasitene. The evidence suggests that petasitene and the genes responsible for its biosynthesis were first found in the genus Chrysanthemum. The present findings provide insights into the composition, formation, and regulation of these bioactive compounds.

The purpose of this article is to explore new functions and experience methods for children’s air purifiers based on the theory of emotional design. The research method is, first, to collect perceptual vocabulary and typical samples, and use the semantic difference method to build a perceptual evaluation table. Secondly, the analysis data is imported into SPSS software for factor analysis and principal component analysis. Establish user demand models for children’s air purifiers through user interviews. Finally, using the hierarchical analysis method to get the three elements of design: shape, function, experience, and then use the three elements of design to guide practice. The significance of the research lies in the use of emotional design methods, which provide new solutions for communication between parents, children and purifiers.

Artemisinin-based combination therapy (ACT) forms the frontline treatment of malaria. Artemisinin, an endoperoxide sesquiterpenoid lactone biosynthesized by Artemisia annua, is the effective medicine that kills malarial parasites. Due to insufficient production of artemisinin for ACT, millions of people lost their lives in past years worldwide. To solve this severe problem, numerous studies have been undertaken to understand artemisinin biosynthesis and to innovate metabolic engineering technology to increase artemisinin yield. Here, we focus on reviewing progresses achieved in understanding biosynthetic pathway, genetic breeding, metabolic engineering, and synthetic biology. Furthermore, based on current knowledge, we discuss multiple fundamental questions and challenges.

Climate change and geography affect all the living organisms. To date, the effects of climate and geographical factors on plant metabolome largely remain open for worldwide and local investigations. In this study, we designed field experiments with tobacco (Nicotiana tabacum) in India versus USA and used untargeted metabolomics to understand the association of two weather factors and two different continental locations with respect to tobacco metabolism. Field research stations in Oxford, North Carolina, USA, and Rajahmundry, Andhra Pradesh India were selected to grow a commercial tobacco genotype (K326) for 2 years. Plant growth, field management, and leaf curing followed protocols standardized for tobacco cultivation. Gas chromatography–mass spectrometry based unbiased profiling annotated 171 non-polar and 225 polar metabolites from cured tobacco leaves. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) showed that two growing years and two field locations played primary and secondary roles affecting metabolite profiles, respectively. PCA and Pearson analysis, which used nicotine, 11 other groups of metabolites, two locations, temperatures, and precipitation, revealed that in North Carolina, temperature changes were positively associated with the profiles of sesquiterpenes, diterpenes, and triterpenes, but negatively associated with the profiles of nicotine, organic acids of tricarboxylic acid, and sugars; in addition, precipitation was positively associated with the profiles of triterpenes. In India, temperature was positively associated with the profiles of benzenes and polycyclic aromatic hydrocarbons, but negatively associated with the profiles of amino acids and sugar. Further comparative analysis revealed that nicotine levels were affected by weather conditions, nevertheless, its trend in leaves was independent of two geographical locations and weather changes. All these findings suggested that climate and geographical variation significantly differentiated the tobacco metabolism.

A large diameter porous SnO2 nanospheres/graphene composite (p-SNG) was synthesized by a one-pot in situ hydrothermal method for the first time. XRD studies and FE-SEM, TEM images indicate that the porous SnO2 nanospheres with diameters of about 200 nm are distributed on the graphene nanosheets (GNS) uniformly. In comparison with the bare SnO2 nanospheres, the as-synthesized p-SNG exhibited enhanced initial charge and discharge capacity, superior cycle performance and rate performance. Electrochemical measurements demonstrated that the p-SNG composite delivered a reversible discharge capacity of 921.5 mA h g−1 at a current density of 400 mA g−1 after 50 cycles and a rate performance of 802.9 mA h g−1 at a high current density of 1600 mA g−1.

Scenarios of genes to metabolites in Artemisia annua remain uninvestigated. Here, we report the use of an integrated approach combining metabolomics, transcriptomics, and gene function analyses to characterize gene-to-terpene and terpene pathway scenarios in a self-pollinating variety of this species. Eighty-eight metabolites including 22 sesquiterpenes (e.g., artemisinin), 26 monoterpenes, two triterpenes, one diterpene and 38 other non-polar metabolites were identified from 14 tissues. These metabolites were differentially produced by leaves and flowers at lower to higher positions. Sequences from cDNA libraries of six tissues were assembled into 18 871 contigs and genome-wide gene expression profiles in tissues were strongly associated with developmental stages and spatial specificities. Sequence mining identified 47 genes that mapped to the artemisinin, non-amorphadiene sesquiterpene, monoterpene, triterpene, 2-C-methyl-D-erythritol 4-phosphate and mevalonate pathways. Pearson correlation analysis resulted in network integration that characterized significant correlations of gene-to-gene expression patterns and gene expression-to-metabolite levels in six tissues simultaneously. More importantly, manipulations of amorpha-4,11-diene synthase gene expression not only affected the activity of this pathway toward artemisinin, artemisinic acid, and arteannuin b but also altered non-amorphadiene sesquiterpene and genome-wide volatile profiles. Such gene-to-terpene landscapes associated with different tissues are fundamental to the metabolic engineering of artemisinin.

The dried roots of Zanthoxylum nitidum (Roxb.) DC have been widely used in Chinese medicine for the treatment of rheumatoid arthritis, toothache, stomach ache, bruises, and snakebites. Alkaloid content, particularly for nitidine chloride (NC), is a crucial indicator of herb quality. Abuse of plant sources of Z. nitidum, such as the usage of NC content not adhering to the Chinese Pharmacopoeia specification, has appeared in herb markets. Thus, a distribution survey was conducted on wild samples from 35 sites grown in 5 provinces, including the evaluation of morphological and phytochemical differences (three alkaloids, one flavonoid, and one lignin). Consequently, the surveyed plants were attributed to four types: three species (type I, II, and III) and one variety (type IV). The NC content of most type I, II, and III plants met quality guidelines, however, all three type IV plants from Guangxi Province exhibited low NC content; the use of plants from that area should be carefully considered. To identify the biosynthetic pathway of NC and other active compounds, nine transcriptomes of type III (root, stem and leaf, three replicates for each tissue) and three of type I (three leaf replicates) were performed to mine candidate unigenes. A set of 505,386 unigenes with an average length of 866 bp were identified. Coexpression analysis of tissue expression patterns were used to narrow down the candidate genes. Consequently, 23 candidate unigenes involved in the alkaloid biosynthesis pathway, 38 unigenes in the lignan biosynthesis pathway, and 16unigenes in the flavonoid biosynthesis pathway were screened. The distribution survey and phytochemical and transcriptome analyses conducted in this study provided valuable insights into resource preservation and the potential of molecular breeding to improve the quality of Z. nitidum products.

In order to improve the efficiency of medical treatment for the elderly during sudden illness and optimize the first-aid service process in nursing homes, this paper uses the SET analysis method to obtain the design gap of the existing nursing-home emergency service system. Taking the elderly with coronary heart disease as an example, focus on their service acceptance and healthy lifestyle. Use the method of user journey map to visualize the daily behavior of the elderly, followed by the SWOT analysis method to obtain the market competitive advantage, and finally establish a coronary heart disease first-aid service system for nursing homes. It is hoped that this study will provide emergency medical service plans for elderly patients with sudden acute illness and provide a reference for the emergency treatment process of other types of diseases.

Objective: To further perfect service design of “rail to air transfer” mode at Beijing Daxing Airport, optimize the transfer flow and improve passengers’ “door to door” transfer experience. Method: Quality function deployment (QFD) method was introduced into the optimization process of service design. Firstly, the user demand for current “rail to air transfer” mode was collected through questionnaire survey and interview method, followed by analysis and classification using KJ method, user demand weight was determined via analytic hierarch process (AHP) method, the correlation between user demand and design elements was quantitatively evaluated through QFD method, and house of quality (HOQ) was constructed to complete the transformation from user demand into design elements. In the end, service contact analysis was implemented according to obtained parameters, the suggestions for improvement were proposed and the service optimization blueprint was constructed. Conclusion: Starting from user demand, QFD method was integrated with service design to optimize and innovate the service design of the existing “rail to air transfer” mode at Daxing Airport. This study has provided a new strategy and method for improving transfer service and user experience.

Amomum villosum, also known as Fructus Amomi, has been used to treat digestive diseases such as abdominal pain, vomiting, and dysentery. Volatile terpenoids are the active metabolites in the essential oil of Fructus Amomi. Nevertheless, downstream genes responsible for activating metabolites biosynthesis in A. villosum still remain unclear. Here, we report the use of an integrative volatile terpenoid profiling and transcriptomics analysis for mining the corresponding genes involved in volatile terpenoid biosynthesis. Ten terpene synthase (TPS) genes were discovered, and two of them were cloned and functionally characterized. AvTPS1 (AvPS: pinene synthase) catalyzed GPP to form α-pinene and β-pinene; AvTPS3 (AvBPPS: bornyl diphosphate synthase) produced bornyl diphosphate as major product and the other three monoterpenoids as minor products. Metabolite accumulation and gene expression pattern combined with AvPS biochemical characterization suggested that AvPS might play a role in biotic defense. On the other hand, the most active ingredient, bornyl acetate, was highly accumulated in seeds and was consistent with the high expression of AvBPPS, which further indicated that AvBPPS is responsible for the biosynthesis of bornyl acetate, the final metabolite of bornyl diphosphate in A. villosum. This study can be used to improve the quality of A. villosum through metabolic engineering, and for the sustainable production of bornyl acetate in heterologous hosts.

4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) catalyzes the last step of the 2-C-methyl-D-erythritol 4- phosphate (MEP) pathway to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP). To date, little is known regarding effects of an increase or a decrease of a HDR expression on terpenoid and other metabolite profiles in plants. In our study, an Artemisia annua HDR cDNA (namely AaHDR1) was cloned from leaves. Expression profiling showed that it was highly expressed in leaves, roots, stems, and flowers with different levels. Green florescence protein fusion and confocal microscope analyses showed that AaHDR1 was localized in chloroplasts. The overexpression of AaHDR1 increased contents of artemisinin, arteannuin B and other sesquiterpenes, and multiple monoterpenes. By contrast, the suppression of AaHDR1 by anti-sense led to opposite results. In addition, an untargeted metabolic profiling showed that the overexpression and suppression altered non-polar metabolite profiles. In conclusion, the overexpression and suppression of AaHDR1 protein level in plastids differentially affect artemisinin and other terpenoid biosynthesis, and alter non-polar metabolite profiles of A. annua. Particularly, its overexpression leading to the increase of artemisinin production is informative to future metabolic engineering of this antimalarial medicine.

Disease spread of Pseudocercospora fijiensis, causal agent of the black Sigatoka disease of banana, depends on ascospores produced through the sexual reproductive cycle. We used phylogenetic analysis to identify P. fijiensis homologs (PKS8-4 and Hybrid8-3) to the PKS4 polyketide synthases (PKS) from Neurospora crassa and Sordaria macrospora involved in sexual reproduction. These sequences also formed a clade with lovastatin, compactin, and betaenone-producing PKS sequences. Transcriptome analysis showed that both the P. fijiensis Hybrid8-3 and PKS8-4 genes have higher expression in infected leaf tissue compared to in culture. Domain analysis showed that PKS8-4 is more similar than Hybrid8-3 to PKS4. pPKS8-4:GFP transcriptional fusion transformants showed expression of GFP in flask-shaped structures in mycelial cultures as well as in crosses between compatible and incompatible mating types. Confocal microscopy confirmed expression in spermagonia in leaf substomatal cavities, consistent with a role in sexual reproduction. A disruption mutant of pks8-4 retained normal pathogenicity on banana, and no differences were observed in growth, conidial production, and spermagonia production. GC-MS profiling of the mutant and wild type did not identify differences in polyketide metabolites, but did identify changes in saturated fatty acid methyl esters and alkene and alkane derivatives. To our knowledge, this is the first report of a polyketide synthase pathway associated with spermagonia.

Asarum sieboldii Miq., one of the three original plants of TCM ASARI RADIX ET RHIZOMA, is a perennial herb distributed in central and eastern China, the Korean Peninsula, and Japan. Methyleugenol has been considered as the most important constituent of Asarum volatile oil, meanwhile asarinin is also employed as the quality control standard of ASARI RADIX ET RHIZOMA in Chinese Pharmacopeia. They both have shown wide range of biological activities. However, little was known about genes involved in biosynthesis pathways of either methyleugenol or asarinin in Asarum plants. In the present study, we performed de novo transcriptome analysis of plant tissues (e.g., roots, rhizomes, and leaves) at different developmental stages. The sequence assembly resulted in 311,597 transcripts from these plant materials, among which 925 transcripts participated in &lsquo, secondary metabolism&rsquo, with particularly up to 20.22% of them falling into phenylpropanoid biosynthesis pathway. The corresponding enzymes belong to seven families potentially encoding phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-monooxygenase (C4H), p-coumarate 3-hydroxylase (C3H), caffeoyl-CoA O-methyltransferase (CCoAOMT), cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD), and eugenol synthase (EGS). Moreover, 5 unigenes of DIR (dirigent protein) and 11 unigenes of CYP719A (719A subfamily of cytochrome P450 oxygenases) were speculated to be involved in asarinin pathway. Of the 15 candidate CADs, four unigenes that possessed high FPKM (fragments per transcript kilobase per million fragments mapped) value in roots were cloned and characterized. Only the recombinant AsCAD5 protein efficiently converted p-coumaryl, coniferyl, and sinapyl aldehydes to their corresponding alcohols, which are key intermediates employed not only in biosynthesis of lignin but also in that of methyleugenol and asarinin. qRT-PCR revealed that AsCAD5 had a high expression level in roots at three developmental stages. Our study will provide insight into the potential application of molecular breeding and metabolic engineering for improving the quality of TCM ASARI RADIX ET RHIZOMA.

We recently characterized a gene-terpene network that is associated with artemisinin biosynthesis in self-pollinated (SP) Artemisia annua, an effective antimalarial plant. We hypothesize that an alteration of gene expression in the network may improve the production of artemisinin and its precursors. In this study, we cloned an isopentenyl pyrophosphate isomerase (IPPI) cDNA, AaIPPI1, from Artemisia annua (Aa). The full-length cDNA encodes a type-I IPPI containing a plastid transit peptide (PTP) at its amino terminus. After the removal of the PTP, the recombinant truncated AaIPPI1 isomerized isopentenyl pyrophosphate (IPP) to dimethyl allyl pyrophosphate (DMAPP) and vice versa. The steady-state equilibrium ratio of IPP/DMAPP in the enzymatic reactions was approximately 1:7. The truncated AaIPPI1 was overexpressed in the cytosol of the SP A. annua variety. The leaves of transgenic plants produced approximately 4% arteannuin B (g g-1 , dry weight, dw) and 0.17-0.25% artemisinin (g g-1 , dw), the levels of which were significantly higher than those in the leaves of wild-type plants. In addition, transgenic plants showed an increase in artemisinic acid production of more than 1% (g g-1 , dw). In contrast, isoprene formation was significantly reduced in transgenic plants. These results provide evidence that overexpression of AaIPPI1 in the cytosol can lead to metabolic alterations of terpenoid biosynthesis, and show that these transgenic plants have the potential to yield high production levels of arteannuin B as a new precursor source for artemisinin.