A novel bHLH gene responsive to low nitrogen positively regulates the biosynthesis of medicinal tropane alkaloids in Atropa belladonna.

Medicinal tropane alkaloids (TAs), including hyoscyamine, anisodamine and scopolamine, are essential anticholinergic drugs specifically produced in several solanaceous plants. Atropa belladonna is one of the most important medicinal plants that produces TAs. Therefore, it is necessary to cultivate new A. belladonna germplasm with the high content of TAs. Here, we found that the levels of TAs were elevated under low nitrogen (LN) condition, and identified a LN-responsive bHLH transcription factor (TF) of A. belladonna (named LNIR) regulating the biosynthesis of TAs. The expression level of LNIR was highest in secondary roots where TAs are synthesized specifically, and was significantly induced by LN. Further research revealed that LNIR directly activated the transcription of hyoscyamine 6ß-hydroxylase gene (H6H) by binding to its promoter, which converts hyoscyamine into anisodamine and subsequently epoxidizes anisodamine to form scopolamine. Overexpression of LNIR upregulated the expression levels of TA biosynthesis genes and consequently led to the increased production of TAs. In summary, we functionally identified a LN-responsive bHLH gene that facilitated the development of A. belladonna with high-yield TAs under the decreased usage of nitrogen fertilizer.

Alteration in the callogenesis, tropane alkaloid formation, and gene expression in Hyoscyamus niger under clinorotation.

This study aimed to investigate the effects of clinorotation induced by 2-D clinostat on the growth, tropane alkaloid production, gene expression, antioxidant capacity, and cellular defense responses in the callus tissue of Hyoscyamus niger. Callus induction was conducted by putting hypocotyl explants in the MS culture medium supplemented with 1 mgL-1 2,4-D and 1 mgL-1 BAP growth regulators. The sub-cultured calli were placed on a clinostat for 0, 3, 7, and 10 days (2.24 × 10-5 g on the edge of the callus ring). Clinorotation significantly increased callus fresh weight, dry weight, protein, carbohydrate, and proline contents compared to the control, and their maximum contents were obtained after 7 and 10 days. H2O2 level enhanced under clinorotation with a 76.3% rise after 10 days compared to control and positively affected the atropine (77.1%) and scopolamine (69.2%) productions. Hyoscyamine 6-beta hydroxylase and putrescine N-methyltransferase gene expression involved in the tropane alkaloid biosynthesis were upregulated markedly with 14.2 and 17.1-folds increase after 10 days of clinorotation, respectively. The expressions of jasmonic acid, mitogen-activated protein kinase, and ethylene-responsive element-binding transcription factor were upregulated, and the activity of peroxidase and catalase showed a 72.7 and 80% rise after 10 days. These findings suggest that microgravity can enhance callogenesis by stimulating the ROS level, which can impact the antioxidant enzymes, tropane alkaloid formation, and gene expression.

A core root bacteria contribute to plant growth and anisodine accumulation of Anisodus tanguticus.

BACKGROUND: Although it is well recognized that core root microorganisms contribute to plant health and productivity, little is known about their role to the accumulation of secondary metabolites. The roots of Anisodus tanguticus, a traditional herbal medication utilized by Tibetan medicine, are rich in tropane alkaloids. We collected wild A. tanguticus populations throughout a 1500 km transect on the Qinghai-Tibetan Plateau. RESULTS: Our results showed that despite sampling at a distance of 1500 km, the root of A. tanguticus selectively recruits core root bacteria. We obtained 102 root bacterial core OTUs, and although their number only accounted for 2.99% of the total, their relative abundance accounted for 73% of the total. Spearman correlation and random forest analyses revealed that the composition of core root microbiomes was related to anisodine contents, aboveground biomass and nitrogen contents of Anisodus tanguticus. Among them, the main role is played by Rhizobacter, Variovorax, Polaromonas, and Mycobacterium genus that are significantly enriched in roots. Functional prediction by FAPROTAX showed that nitrogen-cycling microorganisms and pathogenic bacteria are strongly associated with anisodine contents, aboveground biomass and nitrogen contents of Anisodus tanguticus. CONCLUSIONS: Our findings show that the root selectively recruits core root bacteria and revealed that the core microbiomes and microbial functions potentially contributed to the anisodine contents, aboveground biomass and nitrogen contents of the plant. This work may increase our understanding of the interactions between microorganisms and plants and improve our ability to manage root microbiota to promote sustainable production of herbal medicines.

Effect of silver nanoparticles on tropane alkaloid production of transgenic hairy root cultures of Hyoscyamus muticus L. and their antimicrobial activity.

The utilization of nanotechnology and biotechnology for enhancing the synthesis of plant bioactive chemicals is becoming increasingly common. The hairy root culture technique can be used to increase secondary metabolites such as tropane alkaloids. Agrobacterium was used to induce hairy roots from various explants of Hyoscyamus muticus. The effect of nano-silver particles (AgNPs) at concentrations of 0, 25, 50, 100, and 200 mg/L on tropane alkaloids synthesis, particularly hyoscyamine and scopolamine, was studied in transgenic hairy root cultures. Different types of explants obtained from 10-day-old seedlings of H. muticus were inoculated with two strains of Agrobacterium rhizogenes (15,834 and A4). The antimicrobial activity of an ethanolic extract of AgNPs-induced hairy root cultures of H. muticus was tested. The frequency of hairy roots was higher in hypocotyl, root, leaf, and stem explants treated with A. rhizogenes strain A4 compared to those treated with strain 15,834. In transgenic hairy root cultures, AgNPs application at a concentration of 100 mg/L resulted in the highest total tropane alkaloid production, which exhibited broad-spectrum antimicrobial activity. The study demonstrated the potential of nano-silver as an elicitor for promoting the production of target alkaloids in Hyoscyamus muticus hairy root cultures, which exhibit high biological activity.

Genomic signatures of the evolution of defence against its natural enemies in the poisonous and medicinal plant Datura stramonium (Solanaceae).

Tropane alkaloids and terpenoids are widely used in the medicine and pharmaceutic industry and evolved as chemical defenses against herbivores and pathogens in the annual herb Datura stramonium (Solanaceae). Here, we present the first draft genomes of two plants from contrasting environments of D. stramonium. Using these de novo assemblies, along with other previously published genomes from 11 Solanaceae species, we carried out comparative genomic analyses to provide insights on the genome evolution of D. stramonium within the Solanaceae family, and to elucidate adaptive genomic signatures to biotic and abiotic stresses in this plant. We also studied, in detail, the evolution of four genes of D. stramonium-Putrescine N-methyltransferase, Tropinone reductase I, Tropinone reductase II and Hyoscyamine-6S-dioxygenase-involved in the tropane alkaloid biosynthesis. Our analyses revealed that the genomes of D. stramonium show signatures of expansion, physicochemical divergence and/or positive selection on proteins related to the production of tropane alkaloids, terpenoids, and glycoalkaloids as well as on R defensive genes and other important proteins related with biotic and abiotic pressures such as defense against natural enemies and drought.

Improved tropane alkaloid production and changes in gene expression in hairy root cultures of two Hyoscyamus species elicited by silicon dioxide nanoparticles.

Species of Hyoscyamus are rich sources of medicinally important tropane alkaloids, which have anticholinergic, antispasmodic and sedative effects and are competitive inhibitors of acetylcholine. The application of nanotechnology and nanomaterials for elicitation is rapidly expanding and recent research indicates that silicon dioxide nanoparticles (SiO2 NPs) can be used as an efficient elicitor to increase the production of hyoscyamine and scopolamine in Hyoscyamus species. Thus, in this work, the effect of SiO2 NPs (0, 25, 50, 100 and 200 mg L-1) with two treatment times (24 and 48 h) on the growth rate, total phenol and flavonoid content (TPC, TFC), antioxidant enzyme activity, tropane alkaloid yield and pmt (putrescine N-methyltransferase) and h6h (hyoscyamine 6-hydroxylase) gene expression levels in hairy roots of two Hyoscyamus species (H. reticulatus and H. pusillus) was investigated. The highest TPC and TFC accumulation was obtained in H. reticulatus elicited by SiO2 NPs (100 and 200 mg L-1), respectively, at 24 h of treatment. High-performance liquid chromatography (HPLC) revealed the highest amount of hyoscyamine (140.15 µg g-1 FW) and scopolamine (67.71 µg g-1 FW) accumulated in H. reticulatus transformed roots treated with 100 mg L-1 SiO2 NPs at 24 h, with a respective increase of 1212% and 272% compared to non-treated roots. In H. pusillus, the highest hyoscyamine (7.42 µg g-1 FW) and scopolamine (15.56 µg g-1 FW) production (about 82% and 241% higher, respectively, compared to the lowest amounts) was achieved with 25 and 100 mg L-1 SiO2 NPs, respectively, at 48 h of treatment. Semi-quantitative RT-PCR analysis determined the highest expression level of pmt and h6h genes in H. reticulatus transformed roots supplemented with 100 mg L-1 SiO2 NPs.

Impacts of microbial assemblage and environmental conditions on the distribution of anatoxin-a producing cyanobacteria within a river network.

Blooms of planktonic cyanobacteria have long been of concern in lakes, but more recently, harmful impacts of riverine benthic cyanobacterial mats been recognized. As yet, we know little about how various benthic cyanobacteria are distributed in river networks, or how environmental conditions or other associated microbes in their consortia affect their biosynthetic capacities. We performed metagenomic sequencing for 22 Oscillatoriales-dominated (Cyanobacteria) microbial mats collected across the Eel River network in Northern California and investigated factors associated with anatoxin-a producing cyanobacteria. All microbial communities were dominated by one or two cyanobacterial species, so the key mat metabolisms involve oxygenic photosynthesis and carbon oxidation. Only a few metabolisms fueled the growth of the mat communities, with little evidence for anaerobic metabolic pathways. We genomically defined four cyanobacterial species, all which shared <96% average nucleotide identity with reference Oscillatoriales genomes and are potentially novel species in the genus Microcoleus. One of the Microcoleus species contained the anatoxin-a biosynthesis genes, and we describe the first anatoxin-a gene cluster from the Microcoleus clade within Oscillatoriales. Occurrence of these four Microcoleus species in the watershed was correlated with total dissolved nitrogen and phosphorus concentrations, and the species that contains the anatoxin-a gene cluster was found in sites with higher nitrogen concentrations. Microbial assemblages in mat samples with the anatoxin-a gene cluster consistently had a lower abundance of Burkholderiales (Betaproteobacteria) species than did mats without the anatoxin-producing genes. The associations of water nutrient concentrations and certain co-occurring microbes with anatoxin-a producing Microcoleus motivate further exploration for their roles as potential controls on the distributions of toxigenic benthic cyanobacteria in river networks.

Sodium nitroprusside stimulated production of tropane alkaloids and antioxidant enzymes activity in hairy root culture of Hyoscyamus reticulatus L.

Hyoscyamus reticulatus L. is a herbaceous biennial belonging to the solanaceae family. Hyoscyamine and scopolamine as main tropane alkaloids accumulated in henbane are widely used in medicine to treat diseases such as parkinson's or to calm schizoid patients. Hairy roots media manipulation which uses elicitors to activate defense mechanisms is one of the main strategies for inducing secondary metabolism as well as increasing the production of valuable metabolites. Cotyledon-derived hairy root cultures were transformed by Agrobacterium rhizogenes. Sodium nitroprusside (SNP), a nitric oxide donor), was used in various concentrations (0, 50, 100, 200 and 300 µM) and exposure times (24 and 48 h). Treatment with SNP led to a significant reduction in fresh and dry weight of hairy roots, compared to control cultures. ANOVA results showed that elicitation of hairy root cultures with SNP at different concentrations and exposure times significantly affected the activity of as antioxidant enzymes such as catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX). The highest hyoscyamine and scopolamine production (about 1.2-fold and 1.5-fold increases over the control) was observed at 50 and 100 µM SNP at 48 and 24 hours of exposure time, respectively. This is the first report of SNP elicitation effects on the production of tropane alkaloids in hairy root cultures.

Targeted expression of Vitreoscilla hemoglobin improves the production of tropane alkaloids in Hyoscyamus niger hairy roots.

Under hypoxic conditions, the expression of Vitreoscilla hemoglobin (VHb) in plants is proposed to increase the productivity of certain oxygen-requiring metabolic pathways by promoting the delivery of oxygen. Tropane alkaloids (TAs) are a class of important plant secondary metabolites with significant medicinal value; the final step in their biosynthesis requires oxygen. Whether heterologous expression of VHb, especially in different subcellular compartments, can accelerate the accumulation of TAs is not known. Herein, the effect of heterologous expression of VHb in different subcellular locations on the TA profile of H. niger hairy roots was investigated. The targeted expression of VHb in the plastids (using pVHb-RecA construct), led to the accumulation of 197.68 µg/g hyoscyamine in the transgenic H. niger hairy roots, which was 1.25-fold of the content present in the lines in which VHb expression was not targeted, and 3.66-fold of that present in the wild type (WT) lines. The content of scopolamine was increased by 2.20- and 4.70-fold in the pVHb-RecA transgenic lines compared to that in the VHb transgenic and WT lines. Our results demonstrate that VHb could stimulate the accumulation of TAs in the transgenic H. niger hairy roots. Quantitative RT-PCR analysis revealed that the expression of key genes involved in TA biosynthesis increased significantly in the VHb transgenic lines. We present the first description of a highly efficient strategy to increase TA content in H. niger. Moreover, our results also shed light on how the production of desired metabolites can be efficiently enhanced by using more accurate and appropriate genetic engineering strategies.

A Phenylpyruvic Acid Reductase Is Required for Biosynthesis of Tropane Alkaloids.

Solanaceous medicinal plants produce tropane alkaloids (TAs). We discovered a novel gene from Atropa belladonna, AbPPAR, which encodes a phenylpyruvic acid reductase required for TA biosynthesis. AbPPAR was specifically expressed in root pericycles and endodermis. AbPPAR was shown to catalyze reduction of phenylpyruvic acid to phenyllactic acid, a precursor of TAs. Suppression of AbPPAR disrupted TA biosynthesis through reduction of phenyllactic acid levels. In summary, we identified a novel enzyme involved in TA biosynthesis.

In Vivo Use of 1D and 2D 1H NMR to Examine the Glycosylation of Scopoletin in Duboisia myoporoides Cell Suspensions.

Cell suspensions initiated from Duboisia myoporoides-a shrub belonging to the Solanaceae family and being a rich source of tropane alkaloids-previously showed their ability to glycosylate scopoletin into scopolin, which represent coumarins showing health benefits. To investigate the time course of this glycosylation reaction, an in vivo NMR approach was developed using a perfusion system in an 8-mm NMR tube and 1H NMR with 1D and 2D (TOCSY and NOESY) experiments. The time course of metabolic changes could therefore be followed without any labeling.

Localization and Organization of Scopolamine Biosynthesis in Duboisia myoporoides R. Br.

Tropane alkaloids (TAs), especially hyoscyamine and scopolamine, are important precursors for anticholinergic and antispasmodic drugs. Hyoscyamine and scopolamine are currently obtained at commercial scale from hybrid crosses of Duboisia myoporoides × Duboisia leichhardtii plants. In this study, we present a global investigation of the localization and organization of TA biosynthesis in a Duboisia myoporoides R. Br. wild-type line. The tissue-specific spatial distribution of TAs within D. myoporoides is presented, including quantification of the TAs littorine, 6-hydroxy hyoscyamine, hyoscyamine, scopolamine and, additionally, hyoscyamine aldehyde as well as scopolamine glucoside. Scopolamine (14.77 ± 5.03 mg g-1), and to a lesser extent hyoscyamine (3.01 ± 1.54 mg g-1) as well as 6-hydroxy hyoscyamine (4.35 ± 1.18 mg g-1), are accumulated in leaves during plant development, with the highest concentration of total TAs detected in 6-month-old plants. Littorine, an early precursor in TA biosynthesis, was present only in the roots (0.46 ± 0.07 mg g-1). During development, the spatial distribution of all investigated alkaloids changed due to secondary growth in the roots. Transcripts of pmt, tr-I and cyp80f1 genes, involved in early stages of TA biosynthesis, were found to be most abundant in the roots. In contrast, the transcript encoding hyoscyamine 6ß-hydroxylase (h6h) was highest in the leaves of 3-month-old plants. This investigation presents the spatial distribution of biochemical components as well as gene expression profiles of genetic factors known to participate in TA biosynthesis in D. myoporoides. The results of this investigation may aid in future breeding or genetic enhancement strategies aimed at increasing the yields of TAs in these medicinally valuable plant species.

Genetically engineered hairy root cultures of Hyoscyamus senecionis and H. muticus: ploidy as a promising parameter in the metabolic engineering of tropane alkaloids.

KEY MESSAGE: Tetraploidy improves overexpression of h6h and scopolamine production of H. muticus, while in H. senecionis, pmt overexpression and elicitation can be used as effective methods for increasing tropane alkaloids. The effects of metabolic engineering in a polyploid context were studied by overexpression of h6h in the tetraploid hairy root cultures of H. muticus. Flow cytometry analysis indicated genetic stability in the majority of the clones, while only a few clones showed genetic instability. Among all the diploid and tetraploid clones, the highest level of h6h transgene expression and scopolamine accumulation was interestingly observed in the tetraploid clones of H. muticus. Therefore, metabolic engineering of the tropane biosynthetic pathway in polyploids is suggested as a potential system for increasing the production of tropane alkaloids. Transgenic hairy root cultures of Hyoscyamus senecionis were also established. While overexpression of pmt in H. senecionis was correlated with a sharp increase in hyoscyamine production, the h6h-overexpressing clones were not able to accumulate higher levels of scopolamine than the leaves of intact plants. Applying methyl jasmonate was followed by a sharp increase in the expression of pmt and a drop in the expression of tropinone reductase II (trII) which consequently resulted in the higher biosynthesis of hyoscyamine and total alkaloids in H. senecionis.

Inhibitor mechanisms in the S1 binding site of the dopamine transporter defined by multi-site molecular tethering of photoactive cocaine analogs.

Dopamine transporter (DAT) blockers like cocaine and many other abused and therapeutic drugs bind and stabilize an inactive form of the transporter inhibiting reuptake of extracellular dopamine (DA). The resulting increases in DA lead to the ability of these drugs to induce psychomotor alterations and addiction, but paradoxical findings in animal models indicate that not all DAT antagonists induce cocaine-like behavioral outcomes. How this occurs is not known, but one possibility is that uptake inhibitors may bind at multiple locations or in different poses to stabilize distinct conformational transporter states associated with differential neurochemical endpoints. Understanding the molecular mechanisms governing the pharmacological inhibition of DAT is therefore key for understanding the requisite interactions for behavioral modulation and addiction. Previously, we leveraged complementary computational docking, mutagenesis, peptide mapping, and substituted cysteine accessibility strategies to identify the specific adduction site and binding pose for the crosslinkable, photoactive cocaine analog, RTI 82, which contains a photoactive azide attached at the 2ß position of the tropane pharmacophore. Here, we utilize similar methodology with a different cocaine analog N-[4-(4-azido-3-I-iodophenyl)-butyl]-2-carbomethoxy-3-(4-chlorophenyl)tropane, MFZ 2-24, where the photoactive azide is attached to the tropane nitrogen. In contrast to RTI 82, which crosslinked into residue Phe319 of transmembrane domain (TM) 6, our findings show that MFZ 2-24 adducts to Leu80 in TM1 with modeling and biochemical data indicating that MFZ 2-24, like RTI 82, occupies the central S1 binding pocket with the (+)-charged tropane ring nitrogen coordinating with the (-)-charged carboxyl side chain of Asp79. The superimposition of the tropane ring in the three-dimensional binding poses of these two distinct ligands provides strong experimental evidence for cocaine binding to DAT in the S1 site and the importance of the tropane moiety in competitive mechanisms of DA uptake inhibition. These findings set a structure-function baseline for comparison of typical and atypical DAT inhibitors and how their interactions with DAT could lead to the loss of cocaine-like behaviors.

[Cloning and characterization of an aromatic amino acid aminotransferase(ArAT) gene involved in tropane alkaloid biosynthesis from Hyoscyamus niger].

Tropane alkaloids are anticholinergic drugs widely used clinically. Biosynthesis of tropane alkaloids in planta involves a step of transamination of phenylalanine. Based on the sequenced transcriptomes of lateral roots and leaves of Hyoscyamus niger, we found three annotated aromatic amino acid aminotransferases, which were respectively named HnArAT1, HnArAT2 and HnArAT3. Sequence analysis showed that HnArAT3 had highest similarity with the reported Atropa belladonna Ab Ar AT4, which was involved in tropane alkaloid(TA) to provide the precursor of the phenyllactic acid moiety. Tissue expression pattern analysis indicated that HnArAT3 was specifically expressed in lateral roots, where is the organ synthesizing tropane alkaloids. Then, method of virus induced gene silencing (VIGS) was used to characterize the function of HnArAT3 in H. niger. Gene expression analysis given by real-time quantitative PCR showed that all the transgenic lines had lower expression levels of HnArAT3 than the non-transgenic control, and HPLC analysis of alkaloids demonstrated significant decrease in the contents of hyoscyamine, anisodamine and scopolamine in planta. These results suggested that HnArAT3 was involved in the phenyllactic acid branch of TA biosynthetic pathway. Molecular cloning and functional identification of HnArAT3 laid the foundation for further understanding of TA biosynthesis and metabolic regulation, and also provided a new candidate gene for engineering biosynthetic pathway of tropane alkaloids.

Potato plants with genetically engineered tropane alkaloid precursors.

MAIN CONCLUSION: Solanum tuberosum tropinone reductase I reduced tropinone in vivo. Suppression of tropinone reductase II strongly reduced calystegines in sprouts. Overexpression of putrescine N -methyltransferase did not alter calystegine accumulation. Calystegines are hydroxylated alkaloids formed by the tropane alkaloid pathway. They accumulate in potato (Solanum tuberosum L., Solanaceae) roots and sprouting tubers. Calystegines inhibit various glycosidases in vitro due to their sugar-mimic structure, but functions of calystegines in plants are not understood. Enzymes participating in or competing with calystegine biosynthesis, including putrescine N-methyltransferase (PMT) and tropinone reductases (TRI and TRII), were altered in their activity in potato plants by RNA interference (RNAi) and by overexpression. The genetically altered potato plants were investigated for the accumulation of calystegines and for intermediates of their biosynthesis. An increase in N-methylputrescine provided by DsPMT expression was not sufficient to increase calystegine accumulation. Overexpression and gene knockdown of StTRI proved that S. tuberosum TRI is a functional tropinone reductase in vivo, but no influence on calystegine accumulation was observed. When StTRII expression was suppressed by RNAi, calystegine formation was severely compromised in the transformed plants. Under phytochamber and green house conditions, the StTRII RNAi plants did not show phenotypic alterations. Further investigation of calystegines function in potato plants under natural conditions is enabled by the calystegine deprived StTRII RNAi plants.

Tropane and Granatane Alkaloid Biosynthesis: A Systematic Analysis.

The tropane and granatane alkaloids belong to the larger pyrroline and piperidine classes of plant alkaloids, respectively. Their core structures share common moieties and their scattered distribution among angiosperms suggest that their biosynthesis may share common ancestry in some orders, while they may be independently derived in others. Tropane and granatane alkaloid diversity arises from the myriad modifications occurring to their core ring structures. Throughout much of human history, humans have cultivated tropane- and granatane-producing plants for their medicinal properties. This manuscript will discuss the diversity of their biological and ecological roles as well as what is known about the structural genes and enzymes responsible for their biosynthesis. In addition, modern approaches to producing some pharmaceutically important tropanes via metabolic engineering endeavors are discussed.

Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids.

Brugmansia arborea is a woody plant species that produces tropane alkaloids (TAs). The gene encoding tropine-forming reductase or tropinone reductase I (BaTRI) in this plant species was functionally characterised. The full-length cDNA of BaTRI encoded a 272-amino-acid polypeptide that was highly similar to tropinone reductase I from TAs-producing herbal plant species. The purified 29kDa recombinant BaTRI exhibited maximum reduction activity at pH 6.8-8.0 when tropinone was used as substrate; it also exhibited maximum oxidation activity at pH 9.6 when tropine was used as substrate. The Km, Vmax and Kcat values of BaTRI for tropinone were 2.65mM, 88.3nkatmg(-1) and 2.93S(-1), respectively, at pH 6.4; the Km, Vmax and Kcat values of TRI from Datura stramonium (DsTRI) for tropinone were respectively 4.18mM, 81.20nkatmg(-1) and 2.40S(-1) at pH 6.4. At pH 6.4, 6.8 and 7.0, BaTRI had a significantly higher activity than DsTRI. Analogues of tropinone, 4-methylcyclohexanone and 3-quinuclidinone hydrochloride, were also used to investigate the enzymatic kinetics of BaTRI. The Km, Vmax and Kcat values of BaTRI for tropine were 0.56mM, 171.62nkat.mg(-1) and 5.69S(-1), respectively, at pH 9.6; the Km, Vmax and Kcat values of DsTRI for tropine were 0.34mM, 111.90nkatmg(-1) and 3.30S(-1), respectively, at pH 9.6. The tissue profiles of BaTRI differed from those in TAs-producing herbal plant species. BaTRI was expressed in all examined organs but was most abundant in secondary roots. Finally, tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were detected in various organs of B. arborea by HPLC. Interestingly, scopolamine constituted most of the tropane alkaloids content in B. arborea, which suggests that B. arborea is a scopolamine-rich plant species. The scopolamine content was much higher in the leaves and stems than in other organs. The gene expression and TAs accumulation suggest that the biosynthesis of hyoscyamine, especially scopolamine, occurred not only in the roots but also in the aerial parts of B. arborea.

The role of nitrogen and phosphorus in regulating Phormidium sp. (cyanobacteria) growth and anatoxin production.

Benthic proliferations of the cyanobacteria Phormidium can cover many kilometres of riverbed. Phormidium can produce neurotoxic anatoxins and ingestion of benthic mats has resulted in numerous animal poisonings in the last decade. Despite this, there is a poor understanding of the environmental factors regulating growth and anatoxin production. In this study, the effects of nitrogen and phosphorus on the growth of two Phormidium strains (anatoxin-producing and non-anatoxin-producing) were examined in batch monocultures. Cell concentrations were significantly reduced under reduced nitrogen (ca. <0.100 mM) and phosphorus conditions (ca. <0.003 mM). Cell concentrations and maximum growth rates were higher for the non-anatoxin-producing strain in all treatments, suggesting there may be an energetic cost to toxin production. Cellular anatoxin concentrations were lowest (169 fg cell(-1)) under the high-nitrogen and high-phosphorus treatment. This supports the growth-differentiation balance hypothesis that suggests actively dividing and expanding cells are less likely to produce secondary-metabolites. Anatoxin quota was highest (>407 fg cell(-1)) in the reduced phosphorus treatments, possibly suggesting that it is produced as a stress response to growth limiting conditions. In all treatments there was a 4-5-fold increase in anatoxin quota in the lag growth phase, possibly indicating it may provide a physiological benefit during initial substrate colonization.

The Securinega alkaloids.

Securinega alkaloids represent a family of plant secondary metabolites known for 50 years. Securinine (1), the most abundant and studied alkaloid of this series was isolated by Russian researchers in 1956. In the following years, French and Japanese scientists reported other Securinega compounds and extensive work was done to elucidate their intriguing structures. The homogeneity of this family relies mainly on its tetracyclic chemical backbone, which features a butenolide moiety (cycle D) and an azabicyclo[3.2.1]octane ring system (rings B and C). Interestingly, after a period of latency of 20 years, the Securinega topic reemerged as a prolific source of new natural structures and to date more than 50 compounds have been identified and characterized. The oligomeric subgroup gathering dimeric, trimeric, and tetrameric units is of particular interest. The unprecedented structure of the Securinega alkaloids was the subject of extensive synthetic efforts culminating in several efficient and elegant total syntheses. The botanical distribution of these alkaloids seems limited to the Securinega, Flueggea, Margaritaria, and Breynia genera (Phyllanthaceae). However, only a limited number of plant species have been considered for their alkaloid contents, and additional phytochemical as well as genetic studies are needed. Concerning the biosynthesis, experiments carried out with radiolabelled aminoacids allowed to identify lysine and tyrosine as the precursors of the piperidine ring A and the CD rings of securinine (1), respectively. Besides, plausible biosynthetic pathways were proposed for virosaine A (38) and B (39), flueggine A (46), and also the different oligomers flueggenine A-D (48-51), fluevirosinine A (56), and flueggedine (20). The case of nirurine (45) and secu'amamine (37) remains elusive and additional studies seem necessary to understand their mode of production. The scope of biological of activities of the Securinega alkaloids was mainly centered on the CNS activity of securinine (1), although the exact mechanism of action remained in part unknown. Nevertheless, for its stimulant and antispasmodic effects securinine nitrate was marketed as a drug in the USSR until the early 1990s. Moreover, securinine (1) and several other Securinega alkaloids recently demonstrated promising anticancer properties. In particular securinine (1) demonstrated markedly benefits in the treatment of acute myeloid leukemia.