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.

Allelopathic Effect of Calotropis procera, Hyoscyamus muticus and Pulicaria undulata Extracts on Seed Germination of Portulaca oleracea and Chenopodium murale.

BACKGROUND AND OBJECTIVE: The use of herbicides leads to severe damage, especially to the environment. The aim of this investigation was operated to study the allelopathic effect of Calotropis procera, Hyoscyamus muticus and Pulicaria undulata aqueous extracts on the seed germination of Portulaca oleracea and Chenopodium murale. MATERIALS AND METHODS: The aerial parts of the plants (Calotropis procera, Hyoscyamus muticus and Pulicaria undulata) washed well in distilled water and were dried in room temperature then ground to fine powder. Aqueous extract was prepared using distilled water. RESULTS: In this study, different concentrations of plant extracts (1, 3 and 5%) were used to illustrate which of these concentrations have the potential to inhibit seed growth of Portulaca oleracea and Chenopodium murale. Concentration of 5% for all plant extracts used to inhibited the growth of all seeds of Chenopodium murale while, concentration of 5% for Pulicaria undulata extract only inhibited the growth of all seeds of Portulaca oleracea. CONCLUSION: This work dealt with the use of allelopathy strategy in preventing the growth of some weeds to be one of the safest alternative ways of using herbicides. The results showed the effective effect of plant extracts for both Calotropis procera, Hyoscyamus muticus and Pulicaria undulata on the seed germination of Portulaca oleracea and Chenopodium murale.

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.

Promoting the accumulation of scopolamine and hyoscyamine in Hyoscyamus niger L. through EMS based mutagenesis.

The overexploitation of medicinal plants is depleting gene pool at an alarming rate. In this scenario inducing the genetic variability through targeted mutations could be beneficial in generating varieties with increased content of active compounds. The present study aimed to develop a reproducible protocol for in vitro multiplication and mutagenesis of Hyoscyamus niger targeting putrescine N-methyltransferase (PMT) and 6ß-hydroxy hyoscyamine (H6H) genes of alkaloid biosynthetic pathway. In vitro raised callus were treated with different concentrations (0.01% - 0.1%) of Ethyl Methane Sulfonate (EMS). Emerging multiple shoots and roots were obtained on the MS media supplemented with cytokinins and auxins. Significant effects on morphological characteristics were observed following exposure to different concentrations of EMS. EMS at a concentration of 0.03% was seen to be effective in enhancing the average shoot and root number from 14.5±0.30 to 22.2 ±0.77 and 7.2±0.12 to 8.8±0.72, respectively. The lethal dose (LD50) dose was calculated at 0.08% EMS. The results depicted that EMS has an intense effect on PMT and H6H gene expression and metabolite accumulation. The transcripts of PMT and H6H were significantly upregulated at 0.03-0.05% EMS compared to control. EMS treated explants showed increased accumulation of scopolamine (0.639 µg/g) and hyoscyamine (0.0344µg/g) compared to untreated.

Phytochemical investigation of Hyoscyamus albus.

The work presented in this paper illustrates the isolation and structure elucidation of secondary metabolites of Hyoscyamus albus. Two new natural source and three known compounds were isolated from the Hyoscyamus albus. Among the isolated compounds, grivilloside H (1) and betulaplatoside (2) were isolated for the first time while scopolamine (3), ß-sitosterol (4) and stigmasterol (5) have been reported previously from the same plant. The structures of all the isolated compounds were established by using modern spectroscopic technique (UV, IR, NMR, and EI-MS) and by comparing with those available in literature.

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.

Mechanisms underlying toxicity and stimulatory role of single-walled carbon nanotubes in Hyoscyamus niger during drought stress simulated by polyethylene glycol.

In this study, seeds of Hyoscyamus niger were exposed to different concentrations (50-800µgmL-1) of single-walled carbon nanotubes (SWCNTs) under different levels of drought stress (0.5-1.5MPa) for 14days. Germinated seeds were subsequently allowed to grow in the same culture media for 7 more days to test the further response of the seedlings in terms of biochemical changes to the employed treatments. Seeds subjected to drought showed reduction in germination percentage, vigor and lengths of roots and shoots. However, inclusion of SWCNTs at the two lowest concentrations significantly alleviated the drought stress (up to moderate levels only)-induced reduction in germination and growth attributes. This happened due to increased water uptake, up-regulation of mechanisms involved in starch hydrolysis, and reduction in oxidative injury indices including H2O2, malondialdehyde contents and electrolyte leakage. The improved plant performance under PEG-induced drought stress was a consequence of changes in the expression of various antioxidant enzymes including SOD, POD, CAT, and APX, and also biosynthesis of proteins, phenolics, and specific metabolites such as proline. Results demonstrate that treatment by low concentrations of SWCNTs can induce tolerance in seedlings against low to moderate levels of drought through enhancing water uptake and activating plant defense system.

Microclimatic variation in UV perception and related disparity in tropane and quinolizidine alkaloid composition of Atropa acuminata, Lupinus polyphyllus and Hyoscyamus niger.

The aim of current research was to evaluate the physiological adjustment in three medicinal herbs viz., Atropa acuminata, Lupinus polyphyllus and Hyoscyamus niger to the winter period characterised by intense UV flux in Kashmir valley across the North Western Himalaya. Quinolizidine (QA) and tropane alkaloid (TA) concentrations were analysed in these herbs thriving at two different altitudes via GC-MS and correlated by PCA analysis. This study investigated the hypothesis that UV reflectance and absorbance at low temperatures are directly related to disparity in alkaloid accumulation. Among QAs in L. polyphyllus, ammodendrine and lupanine accumulated at higher concentration and exhibited significant variation of 186.36% and 95.91% in ammodendrine and lupanine respectively in both sites. Tetrahydrohombifoline displayed non-significant variation of about 9.60% irrespective of sites. Among tropane alkaloid (TA), hyoscyamine was recorded as the most abundant constituent irrespective of the plant and site while apotropine accumulated in lesser quantity in A. acuminata than H. niger. However, apotropine demonstrated significant variation of 175% among both sites. The final concentration of quinolizidine (QA) and tropane alkaloid (TA) reflects the interplay between reflectance and absorbance of UV radiation response field. These findings suggest that spectral response of UV light contributes directly to alkaloid biosynthesis.

Changes in cell size and number and in rhizodermal development contribute to root tip swelling of Hyoscyamus albus roots subjected to iron deficiency.

Root tip swelling is a common phenomenon observed when plant roots are subjected to Fe deficiency. We analysed whether an increase in cell number or an enlargement of cell width was involved in this phenomenon. Root tips of Hyoscyamus albus cultured with or without Fe were stained with fluorescent SYTO14 and analysed by confocal laser-scanning microscopy. Time-course and position-based examination revealed that the inhibition of longitudinal cell elongation and acceleration of transverse cell enlargement under Fe deficiency started from the tips and then extended towards the base during the time-course period. An increase in cell number also occurred behind the tips. In addition, the development of rhizodermal protrusions was observed on the surface of roots subjected to Fe deficiency. These results indicated that changes in cell size and number and in root hair development were all involved in root tip swelling.

Production of tropane alkaloids in Hyoscyamus niger (black henbane) hairy roots grown in bubble-column and spray bioreactors.

Hairy root cultures of Hyoscyamus niger were cultivated in shake-flasks, a bubble-column bioreactor and a hybrid bubble-column/spray bioreactor and evaluated for alkaloid production. The latter gave the highest anisodamine content (0.67 mg/g dry wt) whereas scopolamine, hyoscyamine and cuscohygrine concentrations were highest in the bubble-column reactor (5.3, 1.6 and 26.5 mg/g dry wt, respectively). Both bioreactors gave similar productivities of scopolamine (1 and 0.98 mg/l day) and cuscohygrine (5 and 5.4 mg/l day), but anisodamine productivity was 3.5-fold higher in the hybrid bioreactor (HB) (0.02 and 0.07 mg/l day, respectively). Elicitation with methyl jasmonate increased scopolamine productivity by 146 % in roots grown in the HB whereas their permeabilization with DMSO caused 4-, 5-, 25- and 28-fold increase in scopolamine, hyoscyamine, anisodamine and cuscohygrine concentrations in the growth medium. In situ extraction with Amberlite XAD-2 doubled scopolamine productivity in the hybrid reactor after 50 days.

An atypical pattern of accumulation of scopolamine and other tropane alkaloids and expression of alkaloid pathway genes in Hyoscyamus senecionis.

A cDNA encoding hyoscyamine 6ß-hydroxylase (H6H, EC 1.14.11.11), a bifunctional enzyme catalyzing the last two steps in the scopolamine biosynthetic pathway, was isolated from Hyoscyamus senecionis, a medicinal plant endemic to the Iranian plateau. Expression analysis indicates that Hsh6h is expressed in all tested organs of H. senecionis including roots, rhizomes, leaves, stems and flowers unlike the other tropane alkaloid producing species. In parallel to this, in leaves, levels of scopolamine, the product of H6H, were higher than the substrate hyoscyamine. These data suggest that not only does the conversion of hyoscyamine to scopolamine take place in the root, followed by translocation to aerial parts, but also accumulated hyoscyamine in the aerial parts may be converted to scopolamine by activity of HsH6H. Analysis of expression profiles of putrescine N-methyltransferase and tropinone reductase I and II genes also indicates the organ-independent expression of these genes. Here we also introduce H. senecionis as an important tropane alkaloid producing species with its thick underground parts as a source of hyoscyamine, while its leaves can be considered as a source of scopolamine.

In vitro antibacterial activity and phytochemical analysis of White henbane treated by phytohormones.

This study reported the effect of interaction between cytokinins and auxins to enhance accumulation of alkaloids in White henbane (Hyoscyamus albus L.). Plants of this specie were grown under controlled conditions and treated with plant-hormones: Auxins by: 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 3-Indole Acetic Acid (IAA), Cytokinins by: Kinetin (K) and Benzyl Amino Purine (BAP), at 0-10 and 20 mg L(-1) rates isolated and interacted. The results showed that treatment of 2, 4-D and K at the highest applied rates 20 mg L(-1) increased the accumulation threefold rate estimated to 2.321% in the root plant part and 1.702% in the aerial plant part with the same plant-hormones but dosage of (20x10 mg L(-1)) in order. The TLC for alkaloid extracts shows that H. albus L. contains 6 alkaloids. In this study, it was concluded that the treatment with interaction of (Kx2,4-D) (20x20 mg L(-1)) gives the highest percent of alkaloids in the root and shoot parts compared to plant-hormones separated. The in vitro antibacterial activity was determined on microorganisms: Pseudomonas stutzeri, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae. And performed by disc diffusion assay. Respectively, ethanol extracts showed no inhibitory effect on the microorganisms, however alkaloid extracts of H. albus L. of the same treatments with plant-hormones of shoot and root parts showed antibacterial activity against microorganisms that were tested. The results obtained in the present study suggest that alkaloid of H. albus L. can be used in treating diseases caused by the test organisms.

Increased de novo riboflavin synthesis and hydrolysis of FMN are involved in riboflavin secretion from Hyoscyamus albus hairy roots under iron deficiency.

Riboflavin secretion by Hyoscyamus albus hairy roots under Fe deficiency was examined to determine where riboflavin is produced and whether production occurs via an enhancement of riboflavin biosynthesis or a stimulation of flavin mononucleotide (FMN) hydrolysis. Confocal fluorescent microscopy showed that riboflavin was mainly localized in the epidermis and cortex of the root tip and, at the cellular level, in the apoplast. The expressions of three genes involved in the de novo biosynthesis of riboflavin (GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase; 6,7-dimethyl-8-ribityllumazine synthase; riboflavin synthase) were compared between Fe-starved and Fe-replete roots over a time-course of 7 days, using RT-PCR. All three genes were found to be highly expressed over the period 1-7 days in the roots cultured under Fe deficiency. Since riboflavin secretion began to be detected only from 3 days, there was a lag phase observed between the increased transcript accumulations and riboflavin secretion. To determine whether FMN hydrolysis might contribute to the riboflavin secretion in Fe-deficient root cultures, FMN hydrolase activity was determined and was found to be substantially increased after 3 days, when riboflavin secretion became detectable. These results suggested that not only de novo riboflavin synthesis but also the hydrolysis of FMN contributes to riboflavin secretion under conditions of Fe deficiency. Respiration activity was assayed during the time-course, and was also found to be enhanced after 3 days under Fe deficiency, suggesting a possible link with riboflavin secretion. On the other hand, several respiratory inhibitors were found not to affect riboflavin synthase transcript accumulation.

Production of sesquiterpene-type phytoalexins by hairy roots of Hyoscyamus albus co-treated with cupper sulfate and methyl jasmonate.

The production of sesquiterpene-type phytoalexins with a vetispyradiene skeleton by Hyoscyamus albus hairy roots induced by methyl jasmonate (MeJA) was reported in a previous paper. The production pattern on co-treatment with cupper sulfate and MeJA (CuSO(4)-MeJA) showed a TLC profile differing from that on treatment with MeJA. Thus, we studied the production of phytoalexins on hairy root culture involving co-treatment with CuSO(4)-MeJA. In the experiment, many sesquiterpene-type phytoalexins with a vetispyradiene skeleton were isolated, most of which were different from the products reported in the previous paper. Here, we isolated four new phytoalexins (1-4) along with known compounds 5-10 from the culture medium of H. albus hairy roots co-treated with MeJA-CuSO(4). The structures of the new compounds (1-4) were determined as: (3R,4S,5R,7S,9R)-3-acetoxy-9-(2-methylpropionyloxy)solavetivone (1), (3R,4S,5R,7S,9R)-3-hydroxy-9-(3-methylbutanoyloxy)solavetivone (2), (3R,4S,5R,7S,9R)-3-acetoxy-9-(3-methyl-butanoyloxy)solavetivone (3), and (3R,4S,5R,7S,9R)-3-acetoxy-9-(3-methyl-2-butenoyloxy)-solavetivone (4) based on MS and NMR including 2D-NMR data. These findings indicated that the production of phytoalexins in H. albus hairy roots yielded different products based on treatment with different chemicals (CuSO(4), MeJA, and MeJA-CuSO(4)).

Iron deficiency induces changes in riboflavin secretion and the mitochondrial electron transport chain in hairy roots of Hyoscyamus albus.

Hyoscyamus albus hairy roots secrete riboflavin under Fe-deficient conditions. To determine whether this secretion was linked to an enhancement of respiration, both riboflavin secretion and the reduction of 2,3,5-triphenyltetrazolium chloride (TTC), as a measure of respiration activity, were determined in hairy roots cultured under Fe-deficient and Fe-replete conditions, with or without aeration. Appreciable TTC-reducing activity was detected at the root tips, at the bases of lateral roots and in internal tissues, notably the vascular system. TTC-reducing activity increased under Fe deficiency and this increase occurred in concert with riboflavin secretion and was more apparent under aeration. Riboflavin secretion was not apparent under Fe-replete conditions. In order to examine which elements of the mitochondrial electron transport chain might be involved, the effects of the respiratory inhibitors, barbiturate, dicoumarol, malonic acid, antimycin, KCN and salicylhydroxamic acid (SHAM) were investigated. Under Fe-deficient conditions, malonic acid affected neither root growth, TTC-reducing activity nor riboflavin secretion, whereas barbiturate and SHAM inhibited only root growth and TTC-reducing activity, respectively, and the other compounds variously inhibited growth and TTC-reducing activity. Riboflavin secretion was decreased, in concert with TTC-reducing activity, by dicoumarol, antimycin and KCN, but not by SHAM. In Fe-replete roots, all inhibitors which reduced riboflavin secretion in Fe-deficient roots showed somewhat different effects: notably, antimycin and KCN did not significantly inhibit TTC-reducing activity and the inhibition by dicoumarol was much weaker in Fe-replete roots. Combined treatment with KCN and SHAM also revealed that Fe-deficient and Fe-replete roots reduced TTC in different ways. A decrease in the Fe content of mitochondria in Fe-deficient roots was confirmed. Overall, the results suggest that, under conditions of Fe deficiency in H. albus hairy roots, the alternative NAD(P)H dehydrogenases, complex III and complex IV, but not the alternative oxidase, are actively involved both in respiration and in riboflavin secretion.

Responses of fungi to tropane alkaloids produced by a medicinal plant Hyoscyamus muticus (Egyptian henbane).

Antifungal activity of hyoscyamine (Hcy) and scopolamine (Sco) were determined by TLC-bioautography against fungi associated with H. muticus grown in Egypt, and those isolated from other plants grown in Japan. All 40 fungal strains were tolerant to Sco and sensitive to Hcy, exhibiting a growth inhibition zone around the Hcy spot on the bioautography plate. The strains were grouped into three types based on the appearance of the inhibition zone: (i) 17 strains exhibiting a clear inhibition zone, which remained clear at 8 d after incubation (type I); (ii) 22 strains exhibiting the inhibition zone with a brown circle surrounding the zone and regrowth within the inhibition zone (type II); (iii) 1 strain exhibiting the inhibition zone with no brown circle and regrowth within the inhibition zone (type III). In the type II and III strains, Hcy disappeared, and other alkaloids were found in the inhibition zones in its place. Hcy feeding experiments using Penicillium purpurogenum (type II) and Cunninghamella elegans (type III) revealed that these fungi may convert Hcy to a new alkaloid compound.

Root tip-dependent, active riboflavin secretion by Hyoscyamus albus hairy roots under iron deficiency.

Hyoscyamus albus hairy roots with/without an exogenous gene (11 clones) were established by inoculation of Agrobacterium rhizogenes. All clones cultured under iron-deficient condition secreted riboflavin from the root tips into the culture medium and the productivity depended on the number and size of root tips among the clones. A decline of pH was observed before riboflavin production and root development. By studying effects of proton-pump inhibitors, medium acidification with external organic acid, and riboflavin addition upon pH change and riboflavin productivity, we indicate that riboflavin efflux is not directly connected to active pH reduction, and more significantly active riboflavin secretion occurs as a response to an internal requirement in H. albus hairy roots under iron deficiency.

Tropane alkaloids production in transgenic Hyoscyamus niger hairy root cultures over-expressing putrescine N-methyltransferase is methyl jasmonate-dependent.

The cDNA from Nicotiana tabacum encoding Putrescine N-methyltransferase (PMT), which catalyzes the first committed step in the biosynthesis of tropane alkaloids, has been introduced into the genome of a scopolamine-producing Hyoscyamus niger mediated by the disarmed Agrobacterium tumefaciens strain C58C1, which also carries Agrobacterium rhizogenes Ri plasmid pRiA4, and expressed under the control of the CaMV 35S promoter. Hairy root lines transformed with pmt presented fivefold higher PMT activity than the control, and the methylputrescine (MPUT) levels of the resulting engineered hairy roots increased four to fivefold compared to the control and wild-type roots, but there was no significant increase in tropane alkaloids. However, after methyl jasmonate (MeJA) treatment, a considerable increase of PMTase and endogenous H6Hase as well as an increase in scopolamine content was found either in the transgenic hairy roots or the control. The results indicate that hairy root lines over-expressing pmt have a high capacity to synthesize MPUT, whereas their ability to convert hyoscyamine into scopolamine is very limited. Exposure to MeJA strongly stimulated both polyamine and tropane biosynthesis pathways and elicitation led to more or less enhanced production simultaneously.

Heterologous expression of Vitreoscilla hemoglobin (VHb) and cultivation conditions affect the alkaloid profile of Hyoscyamus muticus hairy roots.

Fast-growing hairy root cultures of Hyoscyamus muticus induced by Agrobacterium rhizogenes offer a potential production system for tropane alkaloids. Oxygen deficiency has been shown to limit growth and biomass accumulation of hairy roots, whereas little experimental data is available on the effect of oxygen on alkaloid production. We have investigated the effect of Vitreoscilla hemoglobin (VHb) expression and cultivation conditions on the complete alkaloid profile of H. muticus hairy roots in shake flasks and in a laboratory scale bioreactor. We optimized the growth medium composition and studied the effects of sucrose, ammonium, nitrate, and phosphate on growth and alkaloid production. Maximum biomass accumulation was achieved with the highest and maximum hyoscyamine content with the lowest sucrose concentration. The optimum nitrate concentration for growth was higher for the VHb line than the control. Neither VHb expression nor aeration improved the hyoscyamine content significantly, thus suggesting that hyoscyamine biosynthesis is not limited by oxygen availability. Interestingly, the effect of VHb expression on the alkaloid profile was slightly different from that of aeration. VHb expression did not affect the concentrations of cuscohygrine, which was increased by aeration. Therefore, the effect of VHb is probably not related only to its ability to increase the intracellular effective oxygen concentration.

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures.

Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent. Here, we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme putrescine N-methyltransferase (PMT) and the downstream enzyme hyoscyamine 6 beta-hydroxylase (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscyamus niger) hairy root cultures. Transgenic hairy root lines expressing both pmt and h6h produced significantly higher (P < 0.05) levels of scopolamine compared with the wild-type and transgenic lines harboring a single gene (pmt or h6h). The best line (T(3)) produced 411 mg/liter scopolamine, which was over nine times more than that in the wild type (43 mg/liter) and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H(11) (184 mg/liter). To our knowledge, this is the highest scopolamine content achieved through genetic engineering of a plant. We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield, which was more efficient than plants harboring only one of the two genes. It seems that the pulling force of the downstream enzyme (the faucet enzyme) H6H plays a more important role in stimulating scopolamine accumulation in H. niger whereas the functioning of the upstream enzyme PMT is increased proportionally. This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.