Transcriptomic investigation of the biochemical function of 7-dehydrocholesterol reductase 1 from the traditional Chinese medicinal plant Anemarrhena asphodeloides Bunge.

Anemarrhena asphodeloides Bunge (Liliaceae) is an important Traditional Chinese Medicine herb, which contains up to 6 % total steroidal saponins (timosaponins) and has multiple pharmacological properties. However, the timosaponin biosynthetic pathway has not been extensively investigated. Here we conducted de novo transcriptome sequencing and analysis of A. asphodeloides Bunge and screened for candidate genes involved in the timosaponin biosynthetic pathway. Targeted metabolite analysis showed that timosaponins primarily accumulated in rhizomes, while phytosterols (including cholesterol) were distributed throughout various organs. Most of the identified candidate genes of the timosaponin biosynthetic pathway were also highly expressed in the rhizome, consistent with the results of metabolic analysis. Based on the transcriptome results, two candidate 7-dehydrocholesterol reductase genes were cloned and heterologously expressed in the yeast Saccharomyces cerevisiae. The purified and identified products supported that Aa7DR1 possessed Δ7-reduction activity in yeast and therefore may be involved in the timosaponins biosynthetic pathway in A. asphodeloides Bunge. Phylogenetic analysis showed Aa7DR1 belongs to monocotyledonous Δ7 reductase of phytosterol biosynthesis. These data expand our understanding of timosaponin biosynthesis.

Current Status of Mining, Modification, and Application of Cellulases in Bioactive Substance Extraction.

Cellulases have been used to extract bioactive ingredients from medical plants; however, the poor enzymatic properties of current cellulases significantly limit their application. Two strategies are expected to address this concern: (1) new cellulase gene mining strategies have been promoted, optimized, and integrated, thanks to the improvement of gene sequencing, genomic data, and algorithm optimization, and (2) known cellulases are being modified, thanks to the development of protein engineering, crystal structure data, and computing power. Here, we focus on mining strategies and provide a systemic overview of two approaches based on sequencing and function. Strategies based on protein structure modification, such as introducing disulfide bonds, proline, salt bridges, N-glycosylation modification, and truncation of loop structures, have already been summarized. This review discusses four aspects of cellulase-assisted extraction. Initially, cellulase alone was used to extract bioactive substances, and later, mixed enzyme systems were developed. Physical methods such as ultrasound, microwave, and high hydrostatic pressure have assisted in improving extraction efficiency. Cellulase changes the structure of biomolecules during the extraction process to convert them into effective ingredients with better activity and bioavailability. The combination of cellulase with other enzymes and physical technologies is a promising strategy for future extraction applications.

Molecular cloning and functional analysis of hyoscyamine 6ß-hydroxylase (H6H) in the poisonous and medicinal plant Datura innoxia mill.

Datura innoxia Mill., a traditional Chinese herbal medicine, produces tropane alkaloids such as hyoscyamine and scopolamine. Scopolamine has a larger demand than hyoscyamine due to its stronger pharmacological effects and fewer side reactions. It is extracted from solanaceous plants. However, the content of scopolamine is lower than hyoscyamine in D. innoxia. Hyoscyamine 6ß-hydroxylase (H6H, EC1.14.11.11) is the key enzyme which can catalyze hyoscyamine to form scopolamine. In this study, a cDNA encoding H6H was cloned from D. innoxia roots and named Dih6h. The full-length cDNA is 1413 bp in length with a 1044-bp open reading frame encoding 347 amino acids. The deduced protein sequence of D. innoxia H6H (DiH6H) shared high identity with H6Hs from other plants. The DiH6H was heterologously expressed in Escherichia coli and purified via His-tag affinity technique. The recombinant DiH6H showed activity in transforming hyoscyamine to scopolamine. Despite Dih6h mRNA was detected in various tissues, its levels in roots were higher than that in other tissues. Indeed, scopolamine accumulation was low in roots, but it was very high in aerial parts, especially in flowers and seeds. These observations suggest that scopolamine may be synthesized in the roots and subsequently transported to the aerial parts. To further verify in vivo function of DiH6H, the cDNA of DiH6H was overexpressed in D. innoxia hairy roots. As expected, an increase of scopolamine production was observed in the positive transformants. The results provide a potential strategy for increasing scopolamine yield by metabolic engineering of its biosynthetic pathway in D. innoxia.

Determination of Quercetin and Flavonol Synthase in Boesenbergia rotunda Rhizome.

BACKGROUND AND OBJECTIVE: Flavonols in plants are catalyzed by flavonol synthase (FLS) enzyme. FLS was reported expressed in flowers and fruits, i.e., Dianthus caryophyllus L. (Caryophyllaceae), Petunia hybrida Hort. (Solanaceae), Arabidopsis thaliana L. (Brassicaceae), Citrus unshiu Marc. (Rutaceae). However, none reported about FLS in medicinal plants, particularly those which possess anti-inflammatory activity. This study was aimed to extract and identify FLS in the rhizome of Boesenbergia rotunda (Zingiberaceae) and to determine quercetin in the ethanol extract of the rhizome. MATERIALS AND METHODS: The protein extraction of the rhizome was carried out by employing Laing and Christeller's (2004) and Wang's (2014) methods. The extracted-proteins were separated by using SDS-PAGE, followed by the measurement of FLS intensity by using Gel Analyzer. The FLS-1 of recombinant A. thaliana was employed as the standard. The determination of quercetin in the rhizome was carried out using LC-MS. RESULTS: The FLS occurred as a thick band at 38 kDa with intensity 116-158. The LC chromatogram of the extract indicated a small peak at 7.94 min similar to that of quercetin standard. The MS spectra at 7.94 min indicated that quercetin is present in the B. rotunda rhizome (m/z = 303.0549). The concentration of quercetin in the extract is 0.022% w/v. CONCLUSION: The FLS, an enzyme which plays an important role in producing quercetin, was detected in B. rotunda rhizome planted in Indonesia. As a consequence, quercetin in a small amount, was also quantified in the rhizome of this plant. This report will add a scientific insight of B. rotunda for biological sciences.

[Cloning,expression and characterization of chalcone isomerase from medicinal plant Chinese sumac (Rhus chinensis)].

Flavonoids are a group of secondary metabolites found in plants. They have many pharmacological functions and play an important role in Chinese sumac( Rhus chinensis),which is a well-known traditional Chinese medicinal plant. Chalcone isomerase( CHI,EC 5. 5. 1. 6) is one of the key enzymes in the flavonoids biosynthesis pathway. In this paper,the full-length c DNA sequence encoding the chalcone isomerase from R. chinensis( designated as Rc CHI) was cloned by RT-PCR and rapid-amplification of c DNA Ends( RACE). The Rc CHI c DNA sequence was 1 058 bp and the open reading frame( ORF) was 738 bp. The ORF predicted to encode a 245-amino acid polypeptide. Rc CHI gene contained an intron and two exons. The sequence alignments revealed Rc CHI shared47. 1%-71. 6% identity with the homologues in other plants. Real-time PCR analysis showed that the total flavonoid levels were positively correlated with tissue-specific expressions of Rc CHI mRNA in different tissues. The recombinant protein was successfully expressed in an Escherichia coli strain with the p GEX-6 P-1 vector. In this paper,the CHI gene was cloned and characterized in the family of Anacardiaceae and will help us to obtain better knowledge of the flavonoids biosynthesis of the flavonoid compounds in R. chinensis.

Functional specialization of UDP-glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin, glycyrrhizin.

Glycyrrhizin, a sweet triterpenoid saponin found in the roots and stolons of Glycyrrhiza species (licorice), is an important active ingredient in traditional herbal medicine. We previously identified two cytochrome P450 monooxygenases, CYP88D6 and CYP72A154, that produce an aglycone of glycyrrhizin, glycyrrhetinic acid, in Glycyrrhiza uralensis. The sugar moiety of glycyrrhizin, which is composed of two glucuronic acids, makes it sweet and reduces its side-effects. Here, we report that UDP-glycosyltransferase (UGT) 73P12 catalyzes the second glucuronosylation as the final step of glycyrrhizin biosynthesis in G. uralensis; the UGT73P12 produced glycyrrhizin by transferring a glucuronosyl moiety of UDP-glucuronic acid to glycyrrhetinic acid 3-O-monoglucuronide. We also obtained a natural variant of UGT73P12 from a glycyrrhizin-deficient (83-555) strain of G. uralensis. The natural variant showed loss of specificity for UDP-glucuronic acid and resulted in the production of an alternative saponin, glucoglycyrrhizin. These results are consistent with the chemical phenotype of the 83-555 strain, and suggest the contribution of UGT73P12 to glycyrrhizin biosynthesis in planta. Furthermore, we identified Arg32 as the essential residue of UGT73P12 that provides high specificity for UDP-glucuronic acid. These results strongly suggest the existence of an electrostatic interaction between the positively charged Arg32 and the negatively charged carboxy group of UDP-glucuronic acid. The functional arginine residue and resultant specificity for UDP-glucuronic acid are unique to UGT73P12 in the UGT73P subfamily. Our findings demonstrate the functional specialization of UGT73P12 for glycyrrhizin biosynthesis during divergent evolution, and provide mechanistic insights into UDP-sugar selectivity for the rational engineering of sweet triterpenoid saponins.

The genome of the medicinal plant Andrographis paniculata provides insight into the biosynthesis of the bioactive diterpenoid neoandrographolide.

Andrographis paniculata is a herbaceous dicot plant widely used for its anti-inflammatory and anti-viral properties across its distribution in China, India and other Southeast Asian countries. A. paniculata was used as a crucial therapeutic treatment during the influenza epidemic of 1919 in India, and is still used for the treatment of infectious disease in China. A. paniculata produces large quantities of the anti-inflammatory diterpenoid lactones andrographolide and neoandrographolide, and their analogs, which are touted to be the next generation of natural anti-inflammatory medicines for lung diseases, hepatitis, neurodegenerative disorders, autoimmune disorders and inflammatory skin diseases. Here, we report a chromosome-scale A. paniculata genome sequence of 269 Mb that was assembled by Illumina short reads, PacBio long reads and high-confidence (Hi-C) data. Gene annotation predicted 25 428 protein-coding genes. In order to decipher the genetic underpinning of diterpenoid biosynthesis, transcriptome data from seedlings elicited with methyl jasmonate were also obtained, which enabled the identification of genes encoding diterpenoid synthases, cytochrome P450 monooxygenases, 2-oxoglutarate-dependent dioxygenases and UDP-dependent glycosyltransferases potentially involved in diterpenoid lactone biosynthesis. We further carried out functional characterization of pairs of class-I and -II diterpene synthases, revealing the ability to produce diversified labdane-related diterpene scaffolds. In addition, a glycosyltransferase able to catalyze O-linked glucosylation of andrograpanin, yielding the major active product neoandrographolide, was also identified. Thus, our results demonstrate the utility of the combined genomic and transcriptomic data set generated here for the investigation of the production of the bioactive diterpenoid lactone constituents of the important medicinal herb A. paniculata.

Functional characterization of squalene epoxidase genes in the medicinal plant Tripterygium wilfordii.

Squalene epoxidase, thought to be one of the rate-limiting enzymes in the biosynthetic pathways of both membrane sterols and triterpenes (e.g., celastrol), catalyses the formation of oxidosqualene as the common precursor of sterols and triterpenoids. In this work, we first found five squalene epoxidase genes (TwSEs) from Tripterygium wilfordii. Tissue expression pattern, consistent with methyl jasmonate induction study, showed that TwSEs1-4 were involved in the production of special metabolites. In contrast, TwSE5 showed a different tissue expression pattern and was not induced by methyl jasmonate. To probe the functions of the TwSEs, we first tried using a prokaryotic system by constructing an engineered bacterium, but we failed to detect their products. Next, we used the CRISPR/Cas9 tool to construct an erg1 mutant yeast by knocking out the ERG1 gene of yeast strain BY4741 and then applied this mutant to identify the function of TwSEs. We found that only TwSEs1-4 can functionally complement the erg1 mutant yeast. This study laid the foundation for the heterologous biosynthesis of special metabolites in Tripterygium wilfordii.

Studies on detection and analysis of proteases in leaf extract of medicinally important plants.

ETHNO-PHARMACOLOGICAL RELEVANCE: The whole plant or the extracts obtained from them have long been used as medicine to treat various human diseases and disorders. Notably, those plants endowed with protease activity have been traditionally used as the agents for treating tumors, digestion disorders, swelling, blood coagulation, fibrinolysis and also for immune-modulation. AIM OF THE STUDY: Proteases occupy a pivotal position in enzyme based industries. Plant proteases have been increasingly exploited for pharmaceutical, food, leather and textile processing industries. Earlier investigations have focused on the occurrence of proteases in medicinally unimportant plants. Therefore it has been aimed to study the occurrence of proteolytic enzymes from medicinally important plants establish any correlation exists between protease activity and medicinal use of individual plants. METHODS: Crude extract were obtained from the leaves of 80 different medicinal plants. Tris-HCl buffer was used as the extraction buffer and the supernatants obtained were used for determination of total protein and protease activity using spectrophotometric methods. Qualitative screening for the presence of protease was carried out with agar diffusion method by incorporating the substrate. SDS-PAGE was used to analyse the isoforms of protease and for determination of relative molecular mass. RESULTS: Relatively higher protease activities were observed in the extracts of leaves of Pongamia pinnata (Fabaceae), Wrightia tinctoria (Apocyanaceae) Acalypha indica (Euphorbiaceae), Adhatoda vasica (Acanthaceae) and Curcuma longa (Zingiberaceae). No correlation was found between the total protein content and protease activity in individual plant species. SDS-PAGE analysis indicated the presence of multiple forms of protease of higher molecular weight range in several plant species. We found a strong correlation between the protease activity and medicinal application of the plant CONCLUSION: The present study has unequivocally revealed that the leaves of medicinal plants could serve as excellent sources of proteases which could be exploited for various industrial, food and pharmaceutical applications.

De Novo Assembly and Analysis of Polygonatum sibiricum Transcriptome and Identification of Genes Involved in Polysaccharide Biosynthesis.

Polygonatum sibiricum polysaccharides (PSPs) are used to improve immunity, alleviate dryness, promote the secretion of fluids, and quench thirst. However, the PSP biosynthetic pathway is largely unknown. Understanding the genetic background will help delineate that pathway at the molecular level so that researchers can develop better conservation strategies. After comparing the PSP contents among several different P. sibiricum germplasms, we selected two groups with the largest contrasts in contents and subjected them to HiSeq2500 transcriptome sequencing to identify the candidate genes involved in PSP biosynthesis. In all, 20 kinds of enzyme-encoding genes were related to PSP biosynthesis. The polysaccharide content was positively correlated with the expression patterns of ß-fructofuranosidase (sacA), fructokinase (scrK), UDP-glucose 4-epimerase (GALE), Mannose-1-phosphate guanylyltransferase (GMPP), and UDP-glucose 6-dehydrogenase (UGDH), but negatively correlated with the expression of Hexokinase (HK). Through qRT-PCR validation and comprehensive analysis, we determined that sacA, HK, and GMPP are key genes for enzymes within the PSP metabolic pathway in P. sibiricum. Our results provide a public transcriptome dataset for this species and an outline of pathways for the production of polysaccharides in medicinal plants. They also present more information about the PSP biosynthesis pathway at the molecular level in P. sibiricum and lay the foundation for subsequent research of gene functions.

Molecular cloning of an ester-forming triterpenoid: UDP-glucose 28-O-glucosyltransferase involved in saponin biosynthesis from the medicinal plant Centella asiatica.

Triterpene saponins include bioactive compounds with structures consisting of triterpene aglycones (sapogenins) and one or more sugar moieties linked through acetal or ester glycosidic linkages at one or more sites. Centella asiatica (L.) Urban is a medicinal plant that contains bioactive ursane-type saponins, such as madecassoside and asiaticoside. In this work, glucosylation of triterpenoids in C. asiatica was investigated starting with plant extracts. An enzyme capable of glucosylating asiatic and madecassic acids was partially purified. Proteomics methods and cDNA sequence data were employed as tools to obtain a full-length cDNA clone encoding a glucosyltransferase. The recombinant gene product, UGT73AD1, was functionally expressed in Escherichia coli and purified by immobilized metal-affinity chromatography. Purified recombinant UGT73AD1 was found to have a narrow specificity, glucosylating asiatic and madecassic acids at the C28 carboxyl. mRNA accumulated in all tissues tested (leaves, stems, roots and flowers), with highest expression in leaves. Thus, UGT73AD1 was identified as a triterpenoid carboxylic acid: UDP-glucose 28-O-glucosyltransferase that appears to be involved in saponin biosynthesis in C. asiatica.

Cytochrome P450 Monooxygenase CYP716A141 is a Unique ß-Amyrin C-16ß Oxidase Involved in Triterpenoid Saponin Biosynthesis in Platycodon grandiflorus.

The roots of Platycodon grandiflorus are widely used as a crude drug. The active components include a variety of triterpenoid saponins. Recent studies have revealed that Cyt P450 monooxygenases (P450s) function as triterpene oxidases in triterpenoid saponin biosynthesis in many plant species. However, there have been no reports regarding triterpene oxidases in P. grandiflorus. In this study, we performed transcriptome analysis of three different P. grandiflorus tissues (roots, leaves and petals) using RNA sequencing (RNA-Seq) technology. We cloned six P450 genes that were highly expressed in roots, and classified them as belonging to the CYP716A, CYP716D and CYP72A subfamilies. We heterologously expressed these P450s in an engineered yeast strain that produces ß-amyrin, one of the most common triterpenes in plants. Two of the CYP716A subfamily P450s catalyzed oxidation reactions of the ß-amyrin skeleton. One of these P450s, CYP716A140v2, catalyzed a three-step oxidation reaction at C-28 on ß-amyrin to produce oleanolic acid, a reaction performed by CYP716A subfamily P450s in a variety of plant species. The other P450, CYP716A141, catalyzed the hydroxylation of ß-amyrin at C-16ß. This reaction is unique among triterpene oxidases isolated to date. These results enhance our knowledge of functional variation among CYP716A subfamily enzymes involved in triterpenoid biosynthesis, and provide novel molecular tools for use in synthetic biology to produce triterpenoid saponins with pre-defined structures.

2-Alkylquinolone alkaloid biosynthesis in the medicinal plant Evodia rutaecarpa involves collaboration of two novel type III polyketide synthases.

2-Alkylquinolone (2AQ) alkaloids are pharmaceutically and biologically important natural products produced by both bacteria and plants, with a wide range of biological effects, including antibacterial, cytotoxic, anticholinesterase, and quorum-sensing signaling activities. These diverse activities and 2AQ occurrence in vastly different phyla have raised much interest in the biosynthesis pathways leading to their production. Previous studies in plants have suggested that type III polyketide synthases (PKSs) might be involved in 2AQ biosynthesis, but this hypothesis is untested. To this end, we cloned two novel type III PKSs, alkyldiketide-CoA synthase (ADS) and alkylquinolone synthase (AQS), from the 2AQ-producing medicinal plant, Evodia rutaecarpa (Rutaceae). Functional analyses revealed that collaboration of ADS and AQS produces 2AQ via condensations of N-methylanthraniloyl-CoA, a fatty acyl-CoA, with malonyl-CoA. We show that ADS efficiently catalyzes the decarboxylative condensation of malonyl-CoA with a fatty acyl-CoA to produce an alkyldiketide-CoA, whereas AQS specifically catalyzes the decarboxylative condensation of an alkyldiketide acid with N-methylanthraniloyl-CoA to generate the 2AQ scaffold via C-C/C-N bond formations. Remarkably, the ADS and AQS crystal structures at 1.80 and 2.20 Å resolutions, respectively, indicated that the unique active-site architecture with Trp-332 and Cys-191 and the novel CoA-binding tunnel with Tyr-215 principally control the substrate and product specificities of ADS and AQS, respectively. These results provide additional insights into the catalytic versatility of the type III PKSs and their functional and evolutionary implications for 2AQ biosynthesis in plants and bacteria.

Functional characterization of NES and GES responsible for the biosynthesis of (E)-nerolidol and (E,E)-geranyllinalool in Tripterygium wilfordii.

Triptolide and celastrol, two principal bioactive compounds in Tripterygium wilfordii, are produced from geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate ((E,E)-FPP) through terpenoid biosynthesis pathway. However, little is known about T. wilfordii terpene synthases which could competitively utilize GGPP and (E,E)-FPP as substrates, producing C15 and C20 tertiary alcohols. Here we firstly cloned the genes encoding nerolidol synthase (NES) and geranyllinalool synthases (GES1, GES2), which are responsible for the biosynthesis of (E)-nerolidol and (E,E)-geranyllinalool. In vitro characterization of recombinant TwNES and TwGES1 revealed both were functional enzymes that could catalyze the conversion of (E,E)-FPP and GGPP to (E)-nerolidol and (E,E)-geranyllinalool, which were consistent with the results of yeast fermentation. Biochemical characterization revealed TwNES and TwGES1 had strong dependency for Mg2+, Km and Kcat/Km values of TwNES for (E,E)-FPP were 12.700 µM and 0.029 s-1/µM, and TwGES1 for GGPP were 2.039 µM and 0.019 s-1/µM. Real-time PCR analysis showed the expression levels of NES and GES1 increased by several fold in the suspension cells treated with alamethicin, indicating TwNES and TwGES1 are likely to utilize GGPP and (E,E)-FPP to generate tertiary alcohols as precursor of plant volatiles, which play important roles in the ecological interactions between T. wilfordii and other organisms.

Molecular characterization, expression, and regulation of Gynostemma pentaphyllum squalene epoxidase gene 1.

Gynostemma pentaphyllum (Thunb.) Makino is a perennial medicinal herb widely distributed in China. This herb contains important medicinal components called gypenosides, which belong to dammarane-type triterpenoid saponins. Squalene epoxidase (SE, EC 1.14.99.7) catalyzes the epoxidation of squalene to form oxidosqualene and is a key regulatory enzyme in triterpenoid saponin biosynthesis. In this study, a SE gene designated as GpSE1 was isolated from G. pentaphyllum leaves. The deduced protein sequence of GpSE1 contained two conserved domains involved in the catalytic function of SE. GpSE1 was expressed as inclusion bodies in Escherichia coli cells, and the HIS-tagged recombinant protein was successfully purified and renatured in vitro. Immunofluorescence indicated that the polygonal reticular fluorescence signal of GpSE1 was significantly stronger in young leaves than in mature leaves and rhizomes. This finding is consistent with the tissue-specific expression pattern of GpSE1 and suggests that the young leaves of G. pentaphyllum mainly serve as the active site of gypenoside synthesis. Methyl jasmonate (MeJA) treatment upregulated GpSE1 expression in both the young and mature leaves of G. pentaphyllum, with greater upregulation in young leaves than in mature leaves. However, the expression of GpSE1 was not enhanced continually with the increase in MeJA concentration. Moreover, the GpSE1 expression was maximally regulated in response to 50 µM MeJA but not to 100 µM MeJA. This result indicates that MeJA exerts a concentration-dependent effect on GpSE1 expression.

Examining the transcriptional response of overexpressing anthranilate synthase in the hairy roots of an important medicinal plant Catharanthus roseus by RNA-seq.

BACKGROUND: Clinically important anti-cancer drugs vinblastine and vincristine are solely synthesized by the terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus. Anthranilate synthase (AS) is a rate-limiting enzyme in the TIA pathway. The transgenic C. roseus hairy root line overexpressing a feedback insensitive ASα subunit under the control of an inducible promoter and the ASß subunit constitutively was previously created for the overproduction of TIAs. However, both increases and decreases in TIAs were detected after overexpressing ASα. Although genetic modification is targeted to one gene in the TIA pathway, it could trigger global transcriptional changes that can directly or indirectly affect TIA biosynthesis. In this study, Illumina sequencing and RT-qPCR were used to detect the transcriptional responses to overexpressing AS, which can increase understanding of the complex regulation of the TIA pathway and further inspire rational metabolic engineering for enhanced TIA production in C. roseus hairy roots. RESULTS: Overexpressing AS in C. roseus hairy roots altered the transcription of most known TIA pathway genes and regulators after 12, 24, and 48 h induction detected by RT-qPCR. Changes in the transcriptome of C. roseus hairy roots was further investigated 18 hours after ASα induction and compared to the control hairy roots using RNA-seq. A unigene set of 30,281 was obtained by de novo assembly of the sequencing reads. Comparison of the differentially expressed transcriptional profiles resulted in 2853 differentially expressed transcripts. Functional annotation of these transcripts revealed a complex and systematically transcriptome change in ASαß hairy roots. Pathway analysis shows alterations in many pathways such as aromatic amino acid biosynthesis, jasmonic acid (JA) biosynthesis and other secondary metabolic pathways after perturbing AS. Moreover, many genes in overall stress response were differentially expressed after overexpressing ASα. CONCLUSION: The transcriptomic analysis illustrates overexpressing AS stimulates the overall stress response and affects the metabolic networks in C. roseus hairy roots. The up-regulation of endogenous JA biosynthesis pathway indicates the involvement of JA signal transduction to regulate TIA biosynthesis in ASαß engineered roots and explained why many of the transcripts for TIA genes and regulators are seen to increase with AS overexpression.

Effects of enhanced UV-B radiation on the nutritional and active ingredient contents during the floral development of medicinal chrysanthemum.

The paper mainly studied the effects of enhanced UV-B radiation on the nutritional and active ingredient contents during the floral development of medicinal chrysanthemum. The experiment included two levels of UV-B radiation (0 and 400µWcm(-2)). The contents of hydrogen peroxide (H2O2), anthocyanin, UV-B absorbing compounds, total chlorophyll and carotenoids, and the activities of phenylalanine ammonia lyase enzyme (PAL) and cinnamic acid-4-hydroxylase enzyme (C4H) in flowers significantly decreased with the floral development. However, the contents of soluble sugar, amino acid and total vitamin C in flowers significantly increased with the floral development. The contents of flavonoid and chlorogenic acid were significantly different in the four stages of floral development, and their highest contents were found in the bud stage (stage 2). In the four stages of floral development, enhanced UV-B radiation significantly increased the contents of H2O2, UV-B absorbing compounds, chlorophyll, carotenoids, soluble sugar, amino acid, vitamin C, flavonoid and chlorogenic acid, and the activities of PLA and C4H in flowers. The results indicated that the highest contents of active and nutrient ingredients in flowers were found not to be in the same developmental stages of flowers. Comprehensive analysis revealed that the best harvest stage of chrysanthemum flowers was between the bud stage and the young flower stage (stage 2 and stage 3), which could simultaneously gain the higher contents of active and nutritional ingredients in flowers.

A Picrinine N-Methyltransferase Belongs to a New Family of γ-Tocopherol-Like Methyltransferases Found in Medicinal Plants That Make Biologically Active Monoterpenoid Indole Alkaloids.

Members of the Apocynaceae plant family produce a large number of monoterpenoid indole alkaloids (MIAs) with different substitution patterns that are responsible for their various biological activities. A novel N-methyltransferase involved in the vindoline pathway in Catharanthus roseus showing distinct similarity to γ-tocopherol C-methyltransferases was used in a bioinformatic screen of transcriptomes from Vinca minor, Rauvolfia serpentina, and C. roseus to identify 10 γ-tocopherol-like N-methyltransferases from a large annotated transcriptome database of different MIA-producing plant species (www.phytometasyn.ca). The biochemical function of two members of this group cloned from V. minor (VmPiNMT) and R. serpentina (RsPiNMT) have been characterized by screening their biochemical activities against potential MIA substrates harvested from the leaf surfaces of MIA-accumulating plants. The approach was validated by identifying the MIA picrinine from leaf surfaces of Amsonia hubrichtii as a substrate of VmPiNMT and RsPiNMT. Recombinant proteins were shown to have high substrate specificity and affinity for picrinine, converting it to N-methylpicrinine (ervincine). Developmental studies with V. minor and R. serpentina showed that RsPiNMT and VmPiNMT gene expression and biochemical activities were highest in younger leaf tissues. The assembly of at least 150 known N-methylated MIAs within members of the Apocynaceae family may have occurred as a result of the evolution of the γ-tocopherol-like N-methyltransferase family from γ-tocopherol methyltransferases.

[Cloning and expression analysis of four carotenoid cleavage dioxygenases genes from Pseudostellaria heterophylla].

To investigate the molecular mechanism of quality formation of Pseudostellaria heterophylla, the carotenoid cleavage dioxygenases (CCDs) genes were cloned from the transcriptome database of P. heterophylla, and analyzed them with bioinformatics analysis and expression analysis. The sequence length of four new gene were 1 617, 1 461, 1 746, 1 875 bp, and subsequently, named as PhCCD1,PhNCED2,PhNCED3 and PhCCD4 according to its genetic relationship with Arabidopsis thaliana. The sequence analysis showed that four new gene were all containing REP65 domains and binding sites of ferrous ion, such as histidine, glutamates and aspartates. Analysis phylogeny showed that PhNCED2 and PhNCED3 were the cluster of NCEDs, PhCCD1 and PhCCD4 were the cluster of CCDs. In addition, PhCCD1 and AtCDD1 of Arabidopsis thaliana, PhCCD4 and AtCCD4 of A. thaliana,PhNCED2, PhNCED3 and AtNCED3 of A. thaliana have high similarities. Analysis of real-time fluorescence quantitative showed that PhNCED2 and PhNCED3 were expressed mainly in underground part, the expression quantity of PhNCED2 reached the highest in fibrous root, PhNCED3 keeps higher in phloem and xylem, it may be the key enzymes of ABA biosynthesis genes. Moreover,PhCCD1 and PhCCD4 were expressed mainly in aerial part,the expression quantity of PhCCD1 reached the highest in leaf,PhCCD4 keeps higher in stem and leaf.It may be involved in the biosynthesis of carotenoids for P. heterophylla. The study obtained CDDs gene of P. heterophylla for the first time,this would lay the foundation of developing the response mechanism of P. heterophylla about external stress further,and then exploring the biological approach of quality formation in P. heterophylla.