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Birgit Dräger - One of the best experts on this subject based on the ideXlab platform.
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Potato plants with genetically engineered Tropane Alkaloid precursors
Planta, 2017Co-Authors: Nadine Küster, Sabine Rosahl, Birgit DrägerAbstract: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.
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immunolocalisation of two tropinone reductases in potato solanum tuberosum l root stolon and tuber sprouts
Planta, 2006Co-Authors: Heike Kaiser, Ute Richter, Ronald Keiner, Anja Brabant, Bettina Hause, Birgit DrägerAbstract:Tropinone reductases (TRs) are essential enzymes in the Tropane Alkaloid biosynthesis, providing either tropine for hyoscyamine and scopolamine formation or providing pseudotropine for calystegines. Two cDNAs coding for TRs were isolated from potato (Solanum tuberosum L.) tuber sprouts and expressed in E. coli. One reductase formed pseudotropine, the other formed tropine and showed kinetic properties typical for tropine-forming tropinone reductases (TRI) involved in hyoscyamine formation. Hyoscyamine and tropine are not found in S. tuberosum plants. Potatoes contain calystegines as the only products of the Tropane Alkaloid pathway. Polyclonal antibodies raised against both enzymes were purified to exclude cross reactions and were used for Western-blot analysis and immunolocalisation. The TRI (EC 1.1.1.206) was detected in protein extracts of tuber tissues, but mostly in levels too low to be localised in individual cells. The function of this enzyme in potato that does not form hyoscyamine is not clear. The pseudotropine-forming tropinone reductase (EC 1.1.1.236) was detected in potato roots, stolons, and tuber sprouts. Cortex cells of root and stolon contained the protein; additional strong immuno-labelling was located in phloem parenchyma. In tuber spouts, however, the protein was detected in companion cells.
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tropinone reductases enzymes at the branch point of Tropane Alkaloid metabolism
Phytochemistry, 2006Co-Authors: Birgit DrägerAbstract:Two stereospecific oxidoreductases constitute a branch point in Tropane Alkaloid metabolism. Products of Tropane metabolism are the Alkaloids hyoscyamine, scopolamine, cocaine, and polyhydroxylated norTropane Alkaloids, the calystegines. Both tropinone reductases reduce the precursor tropinone to yield either tropine or pseudotropine. In Solanaceae, tropine is incorporated into hyoscyamine and scopolamine; pseudotropine is the first specific metabolite on the way to the calystegines. Isolation, cloning and heterologous expression of both tropinone reductases enabled kinetic characterisation, protein crystallisation, and structure elucidation. Stereospecificity of reduction is achieved by binding tropinone in the respective enzyme active centre in opposite orientation. Immunolocalisation of both enzyme proteins in cultured roots revealed a tissue-specific protein accumulation. Metabolite flux through both arms of the Tropane Alkaloid pathway appears to be regulated by the activity of both enzymes and by their access to the precursor tropinone. Both tropinone reductases are NADPH-dependent short-chain dehydrogenases with amino acid sequence similarity of more than 50% suggesting their descent from a common ancestor. Putative tropinone reductase sequences annotated in plant genomes other that Solanaceae await functional characterisation.
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Putrescine N-methyltransferase in Solanum tuberosum L., a calystegine-forming plant
Planta, 2006Co-Authors: Olaf Stenzel, Michael Teuber, Birgit DrägerAbstract:Putrescine N -methyltransferase (PMT, EC 2.1.1.53) catalyses the first specific step in the biosynthesis of Tropane and nicotine Alkaloids. Potato ( Solanum tuberosum L.) contains neither nicotine nor the medicinal Tropane Alkaloids hyoscyamine or scopolamine, but calystegines. They are norTropane Alkaloids with glycosidase inhibitory activity. Based on the assumption of calystegine formation by the Tropane Alkaloid pathway, PMT genes and enzymes were investigated in potato. Sprouting tubers contained both N -methylputrescine and PMT activity. Two cDNA clones coding for PMTs were obtained together with a cDNA clone for spermidine synthase (SPDS, EC 2.5.1.16). The pmt sequences resemble those from Nicotiana tabacum (85% identity) and those from Tropane Alkaloid plants, Atropa belladonna (80% identity) and Hyoscyamus niger (79% identity). They are less similar to SPDS of S. tuberosum (66% identity). Expression of pmt1 and spds cDNA in Escherichia coli yielded active enzymes, while pmt2 expression resulted in insoluble protein. Chimera proteins obtained by fusion of fragments of S. tuberosum pmt2 and H. niger pmt were active as PMT, if the initial part of pmt2 was used, indicating that a mutation in the terminal part of the gene caused insolubility of the enzyme. PMT1 was purified after expression in E. coli and proved to be an active N -methyltransferase without SPDS activity. The enzyme was specific for putrescine ( K _M 250 μM) and inhibited by n -butylamine and cadaverine. While spds was transcribed in all plant organs, pmt transcripts were found in small tuber sprouts only. The results confirm that in potato genes and enzymes specific for the Tropane Alkaloid metabolism are expressed and active.
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overexpression of tropinone reductases alters Alkaloid composition in atropa belladonna root cultures
Journal of Experimental Botany, 2005Co-Authors: Ute Richter, Grit Rothe, Annekatrin Fabian, Bettina Rahfeld, Birgit DrägerAbstract:The medicinally applied Tropane Alkaloids hyoscyamine and scopolamine are produced in Atropa belladonna L. and in a small number of other Solanaceae. Calystegines are norTropane Alkaloids that derive from a branching point in the Tropane Alkaloid biosynthetic pathway. In A. belladonna root cultures, calystegine molar concentration is 2-fold higher than that of hyoscyamine and scopolamine. In this study, two tropinone reductases forming a branching point in the Tropane Alkaloid biosynthesis were overexpressed in A. belladonna. Root culture lines with strong overexpression of the transcripts contained more enzyme activity of the respective reductase and enhanced enzyme products, tropine or pseudotropine. High pseudotropine led to an increased accumulation of calystegines in the roots. Strong expression of the tropine-forming reductase was accompanied by 3-fold more hyoscyamine and 5-fold more scopolamine compared with control roots, and calystegine levels were decreased by 30-90% of control. In some of the transformed root cultures, an increase of total Tropane Alkaloids was observed. Thus, transformation with cDNA of tropinone reductases successfully altered the ratio of tropine-derived Alkaloids versus pseudotropine-derived Alkaloids.
Zhihua Liao - One of the best experts on this subject based on the ideXlab platform.
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enhancing Tropane Alkaloid production based on the functional identification of tropine forming reductase in scopolia lurida a tibetan medicinal plant
Frontiers in Plant Science, 2017Co-Authors: Kaihui Zhao, Junlan Zeng, Tengfei Zhao, Haoxing Zhang, Chunxian Yang, Lingjiang Zeng, Min Chen, Zhihua LiaoAbstract:Scopolia lurida, a native herbal plant species in Tibet, is one of the most effective producers of Tropane Alkaloids. However, the Tropane Alkaloid biosynthesis in this plant species of interest has yet to be studied at the molecular, biochemical, and biotechnological level. Here, we report on the isolation and characterization of a putative short chain dehydrogenase (SDR) gene. Sequence analysis showed that SlTRI belonged to the SDR family. Phylogenetic analysis revealed that SlTRI was clustered with the tropine-forming reductases. SlTRI and the other TA-biosynthesis genes, including putrescine N-methyltransferase (SlPMT) and hyoscyamine 6β-hydroxylase (SlH6H), were preferably or exclusively expressed in the S. lurida roots. The tissue profile of SlTRI suggested that this gene might be involved in Tropane Alkaloid biosynthesis. By using GC-MS, SlTRI was shown to catalyze the tropinone reduction to yield tropine, the key intermediate of Tropane Alkaloids. With the purified recombinant SlTRI from E. coli, an enzymatic assay was carried out; its result indicated that SlTRI was a tropine-forming reductase. Finally, the role of SlTRI in promoting the Tropane Alkaloid biosynthesis was confirmed through metabolic engineering in S. lurida. Specifically, hairy root cultures of S. lurida were established to investigate the effects of SlTRI overexpression on Tropane Alkaloid accumulation. In the SlTRI-overexpressing root cultures, the hyoscyamine contents were c. 1.7- to 2.9-fold higher than those in control while their corresponding scopolamine contents were likewise elevated. In summary, this functional identification of SlTRI has provided for a better understanding of Tropane Alkaloid biosynthesis. It also provides a candidate gene for enhancing Tropane Alkaloid biosynthesis in S. lurida via metabolic engineering.
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Cloning and characterization of an aromatic amino acid aminotransferase(ArAT) gene involved in Tropane Alkaloid biosynthesis from Hyoscyamus niger
Yao xue xue bao = Acta pharmaceutica Sinica, 2017Co-Authors: Wei Qiang, Min Chen, Zhihua Liao, Fei Qiu, Xiao-zhong Lan, Xiaoqiang LiuAbstract: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.
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effects of acetylsalicylic acid and uv b on gene expression and Tropane Alkaloid biosynthesis in hairy root cultures of anisodus luridus
Plant Cell Tissue and Organ Culture, 2014Co-Authors: Baifu Qin, Min Chen, Xiao-zhong Lan, Yaxiong Wang, Zhihua LiaoAbstract:Anisodus luridus hairy root cultures were established to test biological effects of acetylsalicylic acid (ASA) and ultraviolet ray-B (UV-B) on gene expression, Tropane Alkaloid (TA) biosynthesis and efflux. The TAs-pathway gene expression was ASA dosage dependant. The expression of PMT, TRI and CYP80F1 showed no significant difference in hairy root cultures in treatment of 0.01 and 0.1 mM ASA, compared with those without ASA treatment; while 0.01 or 0.1 mM ASA slightly upregulated H6H expression. All the four genes including PMT, TRI, CYP80F1 and H6H had a dramatic increase in 1 mM ASA-treated hairy root cultures compared with control. The expressing levels of all the four genes were much significantly higher in 1 mM ASA-treated hairy root cultures than those in 0.01 and 0.1 mM ASA-treated ones. As expected, hairy root cultures treated with 1 mM ASA had the highest capacity of TAs biosynthesis, in which the content of scopolamine and hyoscyamine reached respectively 57.2 and 14.7 μg g−1 DW. Surprisingly, it was found that 1 mM ASA dramatically induced the efflux of scopolamine. In the liquid medium with 1 mM ASA, the content of scopolamine was 153.4 μg flask−1, about 6.2 folds compared with that of control. At the same time, hyoscyamine was detected at trace levels in liquid medium. In the UV-B stressed hairy root cultures, all the four genes had a very strong increase of gene expression that led to more accumulation of scopolamine and lower accumulation of hyoscyamine. Only trace amounts of hyoscyamine and scopolamine were detected in the liquid medium when hairy root cultures were stressed under UV-B, and this suggested that UV-B did not affect TAs efflux.
Eric Gontier - One of the best experts on this subject based on the ideXlab platform.
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Unravelling the architecture and dynamics of Tropane Alkaloid biosynthesis pathways using metabolite correlation networks.
Phytochemistry, 2015Co-Authors: Thi-kieu-oanh Nguyen, Arash Jamali, Arnaud Lanoue, Eric Gontier, Rebecca DauweAbstract:The Tropane Alkaloid spectrum in Solanaceae is highly variable within and between species. Little is known about the topology and the coordination of the biosynthetic pathways leading to the variety of tropine and pseudotropine derived esters in the Alkaloid spectrum, or about the metabolic dynamics induced by Tropane Alkaloid biosynthesis stimulating conditions. A good understanding of the metabolism, including all ramifications, is however necessary for the development of strategies to increase the abundance of pharmacologically interesting compounds such as hyoscyamine and scopolamine. The present study explores the Tropane Alkaloid metabolic pathways in an untargeted approach involving a correlation-based network analysis. Using GC-MS metabolite profiling, the variation and co-variation among Tropane Alkaloids and primary metabolites was monitored in 60 Datura innoxia Mill. individuals, of which half were exposed to Tropane Alkaloid biosynthesis stimulating conditions by co-culture with Agrobacterium rhizogenes. Considerable variation was evident in the relative proportions of the Tropane Alkaloids. Remodeling of the Tropane Alkaloid spectrum under co-culture with A. rhizogenes involved a specific and strong increase of hyoscyamine production and revealed that the accumulation of hyoscyamine, 3-tigloyloxy-6,7-epoxyTropane, and 3-methylbutyryloxyTropane was controlled independently of the majority of Tropane Alkaloids. Based on correlations between metabolites, we propose a biosynthetic origin of hygrine, the order of esterification of certain di-oxygenated Tropanes, and that the rate of acetoxylation contributes to control of hyoscyamine production. Overall, this study shows that the biosynthesis of Tropane Alkaloids may be far more complex and finely controlled than previously expected.
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Tropane Alkaloid profiling of hydroponic Datura innoxia mill. Plants inoculated with Agrobacterium rhizogenes
Phytochemical Analysis, 2010Co-Authors: Alain Hehn, Cyril Jousse, Thi Le Minh Tran, Mohamad Houssam Al Balkhi, Serge Pilard, David Mathiron, Eric GontierAbstract:Introduction - Hydroponics has been shown as a possible way to produce high quality plant biomass with improved phytochemical levels. Nevertheless, effects of plant biotic and abiotic environment can lead to drastic changes and plant growth conditions must be optimised. Objective - To evaluate how much microbes and Agrobacterium rhizogenes TR7 wild strain may affect the Tropane Alkaloid profile in Datura innoxia Mill. plants cultivated in hydroponic conditions. Methodology - Datura innoxia Mill. plants were cultivated in hydroponic with sterile or non-sterile conditions. For half of the non-sterile plants, Agrobacterium rhizogenes TR7 strain was added to the nutrient solution for hydroponics. The Tropane Alkaloid content of leaves and roots was analysed by UFLC/ESI-HRMS and MS/MS. The metabolite profiles were compared using partial least square-discriminant analysis. Results - In sterile conditions, aerial parts contained more scopolamine than the roots. However, the diversity of Tropane Alkaloids was greater in roots. Furthermore, 21 known compounds and four non-elucidated Tropane Alkaloids were found. The Tropane Alkaloid profile was shown to be statistically different between sterile and non-sterile hydroponic conditions. The levels of 3-acetoxy-6-hydroxyTropane and 3'-hydroxylittorine were higher in plants inoculated with A. rhizogenes. Five other Tropane compounds were found in higher amounts in non-axenic control plants. Hyoscyamine and scopolamine total contents were much higher in the whole plant co-cultivated with A. rhizogenes TR7 than in controls. Furthermore, the leaves and roots of axenic plants contained more Alkaloids than non-sterile ones. Conclusion - In hydroponic conditions, microbes induced variations of the phytochemical levels. Addition of A. rhizogenes TR7 into the nutrient solutions improved the total hyoscyamine and scopolamine production.
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influence of minerals and elicitation on datura stramonium l Tropane Alkaloid production modelization of the in vitro biochemical response
Plant Science, 2009Co-Authors: R Amdoun, Majda Khelifislaoui, Lakhdar Khelifi, S Amroune, Elhadi Benyoussef, Dao Vu Thi, Corinne Assafducrocq, Eric GontierAbstract:Abstract Secondary metabolites are mostly involved in the relationship of the organism with its environment, e.g. in plant defence mechanisms or as signal compound; their synthesis is often induced by different kinds of biotic and/or abiotic stresses. In general the major strategies to improve their productivity are: the optimization of growth and production media or induction of secondary metabolite pathways by elicitors and metabolic engineering. From this point of view, elicitation is largely employed as a strategy to mimic stress effects, which then lead to a physiological response involving secondary metabolite synthesis and accumulation into the hairy root (HR). Thus, studies on the response to elicitation for HR in accordance with nutritional status are necessary. In the present study, which focuses on Tropane Alkaloid production in Datura stramonium L. hairy roots, the effects of minerals in the B5 culture medium [O.L. Gamborg, R.A. Miller, K. Ojima, Nutrient requirements of suspension cultures of soybean root cells, Exp. Cell Res. 50 (1968) 151–158] and elicitor (jasmonic acid) are studied using a mathematical model approach (screening analysis) and factorial design 23 was carried out to study the response to elicitation in accordance with nutritional status of HR for [NO3−], [H2PO4−] and [Ca2+]. The modelization of different minerals effects showed a response to elicitation only after the 8th day of cultivation. The study of models coefficients revealed that nitrate [NO3−], phosphorus [H2PO4−] and calcium [Ca2+] levels and combinations [NO3−/Ca2+] and [NO3−/H2PO4−/Ca2+] were positive with elicitation; however, interaction effects [NO3−/H2PO4−] and [H2PO4−/Ca2+] were negative. The coefficients position on the 28th day of cultivation, with the Pareto chart allowed to adjust the mathematical model by eliminating the no significant effects [NO3−/H2PO4−], [H2PO4−/Ca2+] and [NO3−/H2PO4−/Ca2+]. As a result, we show that simulation with the adjusted model allows predicting the response to elicitation in accordance with the nutritional state of HR in nitrate and calcium. The measured values on the 28th day were statistically identical with calculated values obtained by mathematical models. These results revealed that HR, with limited nitrate, showed lower response to elicitation by jasmonic acid. Moreover, the adjusted model, of first degree, was not predictably beyond the experimental domain. As a general conclusion, NO3− (62.5 mM) must be added to the B5 medium at the beginning of the culture. The lack of these compounds reduced Alkaloid metabolism, even in combination with elicitor treatments like jasmonic acid. The same trend was observed with Ca2+ (11.1 mM), H2PO4− (6.06 mM) and nitrate/calcium ratio (62.5 mM/11.1 mM), which also increased Alkaloid production when combined with jasmonic acid 10 mM. These positive effects appeared at the 2nd week after initiation of the culture.
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Effects of calcium, alginate, and calcium-alginate immobilization on growth and Tropane Alkaloid levels of a stable suspension cell line of Datura innoxia Mill.
Plant cell reports, 1994Co-Authors: Eric Gontier, B. S. Sangwan, J. N. BarbotinAbstract:A stabilized two-year old suspension of a Datura innoxia cell line, producing small amounts of Tropane Alkaloids (scopolamine and hyoscyamine) was used in this study. Calcium alginate immobilization has been shown to be able to increase secondary metabolite (i. e. Alkaloid) production. The effects of calcium and ungellified alginate were both beneficial for Tropane Alkaloid synthesis; a 10mM calcium chloride supply gave the best results, with a 10-fold yield increase.
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Tropane Alkaloid levels in the leaves of micropropagated datura innoxia plants
Planta Medica, 1993Co-Authors: Eric Gontier, J. N. Barbotin, M A Fliniaux, B S SangwannorrelAbstract:Three androgenic DATURA INNOXIA Mill. (Solanaceae) plants which had been characterised in regard to their Tropane Alkaloid levels were micropropagated through axillary buds. The Alkaloid contents in the leaves of more than 300 micropropagated plants were investigated by ELISA and HPLC, after one and two culture cycles and compared to those of one of seedlings obtained from the same original line from which the androgenic plants had been produced. The plant with the higher Alkaloid level yielded the population with the most Alkaloids. After a second cycle of micropropagation, all the populations contained more Alkaloids than those they had been derived from. All the micropropagated plant populations contained higher levels of Alkaloids than plants grown from seeds.
Koichiro Shimomura - One of the best experts on this subject based on the ideXlab platform.
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Responses of Transformed Root Culture of Atropa belladonna to Salicylic Acid Stress
Journal of Bioscience and Bioengineering, 2002Co-Authors: Kung-ta Lee, Hiroshi Hirano, Takashi Yamakawa, Tohru Kodama, Yasuo Igarashi, Koichiro ShimomuraAbstract:The effect of salicylic acid (SA) on Tropane Alkaloid production and the responses to SA stress of transformed root cultures of Atropa belladonna (belladonna) were investigated. Treatment of A. belladonna transformed roots with 0.2 mM SA did not have any effect on Tropane Alkaloid production, but two compounds were produced in the medium. These were identified as the SA derivatives methylsalicylate and methyl-o-methoxybenzoate by high-resolution mass spectrometry and UV spectrometry. In contrast, treatment with 2 mM salicylic acid stimulated Tropane Alkaloid release from the transformed roots into the medium by up to 35% of the total Alkaloids after 24 h, and the SA derivatives were not observed in the medium. These results revealed that transformed root of A. belladonna exhibits distinct by different responses to SA stress depending on the SA concentration.
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Effects of phytosulfokine-α on growth and Tropane Alkaloid production in transformed roots of Atropa belladonna
Plant Growth Regulation, 2002Co-Authors: Kazuo Sasaki, Koichiro Shimomura, Tatsuaki Ishise, Toshihiro Kobayashi, Yoshikatsuo Matsubayashi, Youji Sakagami, Hironori Umetsu, Hiroshi KamadaAbstract:Phytosulfokine (PSK)-α is a sulphated pentapeptide, isolated fromthe medium of cultured Asparagus officinalis mesophyllcells, that promotes cell proliferation. It is a putative key factor inconditioned medium required for the growth of low-density plant cell cultures.The present study investigates the effect of PSK-α on growth and TropaneAlkaloid production in Atropa belladonna hairy rootstransformed with Agrobacteriumrhizogenes (MAFF 03-01724). Although the growth rates ofhairy roots cultured in medium with orwithout PSK-α for 4 weeks did not show any differences, the productivityof Tropane Alkaloids, especially of hyoscyamine, was enhanced by10−7 or 10−8 M PSK-α. Inaddition, the content of Tropane Alkaloids in transformed roots treated withPSK-α was 1.4 times higher than that of untreated roots after 4 weeks ofculture. The time course of growth and Tropane Alkaloid production inAtropa belladonna transformed roots suggested thatPSK-α influenced the growth of transformed roots during the activegrowingphase, but not Tropane Alkaloid production.
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Genetic Transformation of Atropa belladonna
Transgenic Medicinal Plants, 1999Co-Authors: Mondher El Jaziri, Kayo Yoshimatsu, Koichiro ShimomuraAbstract:Leaves of Atropa belladonna L. (Solanaceae) are mainly used as a source for the Tropane Alkaloids, hyoscyamine and scopolamine, which are not easily produced by chemical synthesis. Thus because of its high morphogenetic potential, the in vitro culture of A. belladonna has been actively investigated as an alternative to field cultivation and as a possible source for Tropane Alkaloid production (for review see Bajaj and Simola 1991) Several studies have confirmed that the product levels in unorganized A. belladonna callus and suspension cultures are generally very low and that root morphology is required for synthesis of Tropane Alkaloid in vitro. However, molecular genetic technology is now available to achieve transformation and regeneration of transgenic A. belladonna plants in which morphological traits and/or particular metabolic step(s) are artificially modified. The hairy root system is demonstrated to be much more genetically stable than dedifferentiated normal (callus) or transformed (crown gall) plant cells. In this chapter some important recent studies related to the genetic transformation of A. belladonna are discussed.
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Effects of aeration on Tropane Alkaloid production by transformed root of Atropa belladonna in flask cultures
Journal of Fermentation and Bioengineering, 1998Co-Authors: Kung-ta Lee, Takashi Yamakawa, Tohru Kodama, Yasuo Igarashi, Koichiro ShimomuraAbstract:Abstract Transformed roots of Atropa belladonna strain M8 were cultured in shaking flasks aerated with normal air at 1 vvm, capped with aluminum foil, or capped with a silicon rubber stopper, to investigate the effects of aeration on root growth and on Tropane Alkaloid production and composition. After four weeks, the Tropane Alkaloid productivity of roots grown in flasks aerated at 1 vvm reached 52.9 mg/ l , and the individual Tropane Alkaloid contents in the roots were, in mg/ l dry wt., (−)-hyoscyamine 4.8, 6β-hydroxyhyoscyamine 1.4, littorine 1.2, and scopolamine 1.0. These results indicate that aeration at 1 vvm air has an important effect on the control of the Tropane Alkaloid composition in root cultures. In particular, the scopolamine ratio increased from 5.7 to 11.9% of the total Alkaloids accumulated in the belladonna cultured roots.
Min Chen - One of the best experts on this subject based on the ideXlab platform.
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enhancing Tropane Alkaloid production based on the functional identification of tropine forming reductase in scopolia lurida a tibetan medicinal plant
Frontiers in Plant Science, 2017Co-Authors: Kaihui Zhao, Junlan Zeng, Tengfei Zhao, Haoxing Zhang, Chunxian Yang, Lingjiang Zeng, Min Chen, Zhihua LiaoAbstract:Scopolia lurida, a native herbal plant species in Tibet, is one of the most effective producers of Tropane Alkaloids. However, the Tropane Alkaloid biosynthesis in this plant species of interest has yet to be studied at the molecular, biochemical, and biotechnological level. Here, we report on the isolation and characterization of a putative short chain dehydrogenase (SDR) gene. Sequence analysis showed that SlTRI belonged to the SDR family. Phylogenetic analysis revealed that SlTRI was clustered with the tropine-forming reductases. SlTRI and the other TA-biosynthesis genes, including putrescine N-methyltransferase (SlPMT) and hyoscyamine 6β-hydroxylase (SlH6H), were preferably or exclusively expressed in the S. lurida roots. The tissue profile of SlTRI suggested that this gene might be involved in Tropane Alkaloid biosynthesis. By using GC-MS, SlTRI was shown to catalyze the tropinone reduction to yield tropine, the key intermediate of Tropane Alkaloids. With the purified recombinant SlTRI from E. coli, an enzymatic assay was carried out; its result indicated that SlTRI was a tropine-forming reductase. Finally, the role of SlTRI in promoting the Tropane Alkaloid biosynthesis was confirmed through metabolic engineering in S. lurida. Specifically, hairy root cultures of S. lurida were established to investigate the effects of SlTRI overexpression on Tropane Alkaloid accumulation. In the SlTRI-overexpressing root cultures, the hyoscyamine contents were c. 1.7- to 2.9-fold higher than those in control while their corresponding scopolamine contents were likewise elevated. In summary, this functional identification of SlTRI has provided for a better understanding of Tropane Alkaloid biosynthesis. It also provides a candidate gene for enhancing Tropane Alkaloid biosynthesis in S. lurida via metabolic engineering.
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Enhancing Tropane Alkaloid Production Based on the Functional Identification of Tropine-Forming Reductase in Scopolia lurida, a Tibetan Medicinal Plant
Frontiers Media S.A., 2017Co-Authors: Kaihui Zhao, Junlan Zeng, Tengfei Zhao, Haoxing Zhang, Chunxian Yang, Lingjiang Zeng, Fei Qiu, Xiaoqiang Liu, Min ChenAbstract:Scopolia lurida, a native herbal plant species in Tibet, is one of the most effective producers of Tropane Alkaloids. However, the Tropane Alkaloid biosynthesis in this plant species of interest has yet to be studied at the molecular, biochemical, and biotechnological level. Here, we report on the isolation and characterization of a putative short chain dehydrogenase (SDR) gene. Sequence analysis showed that SlTRI belonged to the SDR family. Phylogenetic analysis revealed that SlTRI was clustered with the tropine-forming reductases. SlTRI and the other TA-biosynthesis genes, including putrescine N-methyltransferase (SlPMT) and hyoscyamine 6β-hydroxylase (SlH6H), were preferably or exclusively expressed in the S. lurida roots. The tissue profile of SlTRI suggested that this gene might be involved in Tropane Alkaloid biosynthesis. By using GC-MS, SlTRI was shown to catalyze the tropinone reduction to yield tropine, the key intermediate of Tropane Alkaloids. With the purified recombinant SlTRI from Escherichiacoli, an enzymatic assay was carried out; its result indicated that SlTRI was a tropine-forming reductase. Finally, the role of SlTRI in promoting the Tropane Alkaloid biosynthesis was confirmed through metabolic engineering in S. lurida. Specifically, hairy root cultures of S. lurida were established to investigate the effects of SlTRI overexpression on Tropane Alkaloid accumulation. In the SlTRI-overexpressing root cultures, the hyoscyamine contents were 1.7- to 2.9-fold higher than those in control while their corresponding scopolamine contents were likewise elevated. In summary, this functional identification of SlTRI has provided for a better understanding of Tropane Alkaloid biosynthesis. It also provides a candidate gene for enhancing Tropane Alkaloid biosynthesis in S. lurida via metabolic engineering
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Cloning and characterization of an aromatic amino acid aminotransferase(ArAT) gene involved in Tropane Alkaloid biosynthesis from Hyoscyamus niger
Yao xue xue bao = Acta pharmaceutica Sinica, 2017Co-Authors: Wei Qiang, Min Chen, Zhihua Liao, Fei Qiu, Xiao-zhong Lan, Xiaoqiang LiuAbstract: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.
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effects of acetylsalicylic acid and uv b on gene expression and Tropane Alkaloid biosynthesis in hairy root cultures of anisodus luridus
Plant Cell Tissue and Organ Culture, 2014Co-Authors: Baifu Qin, Min Chen, Xiao-zhong Lan, Yaxiong Wang, Zhihua LiaoAbstract:Anisodus luridus hairy root cultures were established to test biological effects of acetylsalicylic acid (ASA) and ultraviolet ray-B (UV-B) on gene expression, Tropane Alkaloid (TA) biosynthesis and efflux. The TAs-pathway gene expression was ASA dosage dependant. The expression of PMT, TRI and CYP80F1 showed no significant difference in hairy root cultures in treatment of 0.01 and 0.1 mM ASA, compared with those without ASA treatment; while 0.01 or 0.1 mM ASA slightly upregulated H6H expression. All the four genes including PMT, TRI, CYP80F1 and H6H had a dramatic increase in 1 mM ASA-treated hairy root cultures compared with control. The expressing levels of all the four genes were much significantly higher in 1 mM ASA-treated hairy root cultures than those in 0.01 and 0.1 mM ASA-treated ones. As expected, hairy root cultures treated with 1 mM ASA had the highest capacity of TAs biosynthesis, in which the content of scopolamine and hyoscyamine reached respectively 57.2 and 14.7 μg g−1 DW. Surprisingly, it was found that 1 mM ASA dramatically induced the efflux of scopolamine. In the liquid medium with 1 mM ASA, the content of scopolamine was 153.4 μg flask−1, about 6.2 folds compared with that of control. At the same time, hyoscyamine was detected at trace levels in liquid medium. In the UV-B stressed hairy root cultures, all the four genes had a very strong increase of gene expression that led to more accumulation of scopolamine and lower accumulation of hyoscyamine. Only trace amounts of hyoscyamine and scopolamine were detected in the liquid medium when hairy root cultures were stressed under UV-B, and this suggested that UV-B did not affect TAs efflux.
