Alkaloid Metabolism In Plants Research Articles

Identification of structurally diverse alkaloids in Corydalis species by liquid chromatography/electrospray ionization tandem mass spectrometry

Alkaloids with significant therapeutic effects are the main active constituents of Corydalis (C.) species. There are several kinds of alkaloids in C. species associated with diverse alkaloid metabolism in plants, but they are rarely identified. This study aimed to identify diverse alkaloids in C. species by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). Several types of alkaloids were extracted from C. species using ultrasonication with 70% CH(3)OH, and the extract was partitioned at pH 2 and 12. Separation of alkaloids was achieved by C18 high-performance liquid chromatography (HPLC), and MS/MS analysis was conducted by electrospray ionization triple-quadrupole mass spectrometry. For further confirmation, LC/Fourier transform ion cyclotron resonance (FTICR)-MS was used to obtain accurate mass data and gas chromatography (GC)/MS combined with trimethylsilyl derivatization was applied for identification of the minor alkaloids. Thirty-three alkaloids among three different C. species were successfully separated and identified by LC/ESI-MS/MS and LC/FTICR-MS. Structural assignment of individual alkaloids was performed according to MS/MS spectral patterns. For further confirmation, accurate mass data of alkaloids by LC/FTICR-MS were obtained within 5 ppm and the GC/MS data for the trimethylsilyl alkaloids were also obtained. Among 33 alkaloids identified from this study, 13 alkaloids were reported for the first time in the investigated C. species. The LC/ESI-MS/MS technique was effective in obtaining structural information and yielded diagnostic ions for diverse alkaloids. Based on the identified 33 alkaloids, marker compounds were suggested for the three C. species with different geographic origins. This study may also be useful for elucidating unknown alkaloids in herbal medicines.

Quantitative 1H nuclear magnetic resonance metabolite profiling as a functional genomics platform to investigate alkaloid biosynthesis in opium poppy.

Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a versatile model system to study plant alkaloid metabolism. The plant is widely cultivated as the only commercial source of the narcotic analgesics morphine and codeine. Variations in plant secondary metabolism as a result of genetic diversity are often associated with perturbations in other metabolic pathways. As part of a functional genomics platform, we used (1)H nuclear magnetic resonance (NMR) metabolite profiling for the analysis of primary and secondary metabolism in opium poppy. Aqueous and chloroform extracts of six different opium poppy cultivars were subjected to chemometric analysis. Principle component analysis of the (1)H NMR spectra for latex extracts clearly distinguished two varieties, including a low-alkaloid variety and a high-thebaine, low-morphine cultivar. Distinction was also made between pharmaceutical-grade opium poppy cultivars and a condiment variety. Such phenotypic differences were not observed in root extracts. Loading plots confirmed that morphinan alkaloids contributed predominantly to the variance in latex extracts. Quantification of 34 root and 21 latex metabolites, performed using Chenomx NMR Suite version 4.6, showed major differences in the accumulation of specific alkaloids in the latex of the low-alkaloid and high-thebaine, low-morphine varieties. Relatively few differences were found in the levels of other metabolites, indicating that the variation was specific for alkaloid metabolism. Exceptions in the low-alkaloid cultivar included an increased accumulation of the alkaloid precursor tyramine and reduced levels of sucrose, some amino acids, and malate. Real-time polymerase chain reaction analysis of 42 genes involved in primary and secondary metabolism showed differential gene expression mainly associated with alkaloid biosynthesis. Reduced alkaloid levels in the condiment variety were associated with the reduced abundance of transcripts encoding several alkaloid biosynthetic enzymes.

Opium poppy and Madagascar periwinkle: model non‐model systems to investigate alkaloid biosynthesis in plants

Alkaloids represent a large and diverse group of compounds that are related by the occurrence of a nitrogen atom within a heterocyclic backbone. Unlike other types of secondary metabolites, the various structural categories of alkaloids are unrelated in terms of biosynthesis and evolution. Although the biology of each group is unique, common patterns have become apparent. Opium poppy (Papaver somniferum), which produces several benzylisoquinoline alkaloids, and Madagascar periwinkle (Catharanthus roseus), which accumulates an array of monoterpenoid indole alkaloids, have emerged as the premier organisms used to study plant alkaloid metabolism. The status of these species as model systems results from decades of research on the chemistry, enzymology and molecular biology responsible for the biosynthesis of valuable pharmaceutical alkaloids. Opium poppy remains the only commercial source for morphine, codeine and semi-synthetic analgesics, such as oxycodone, derived from thebaine. Catharanthus roseus is the only source for the anti-cancer drugs vinblastine and vincristine. Impressive collections of cDNAs encoding biosynthetic enzymes and regulatory proteins involved in the formation of benzylisoquinoline and monoterpenoid indole alkaloids are now available, and the rate of gene discovery has accelerated with the application of genomics. Such tools have allowed the establishment of models that describe the complex cell biology of alkaloid metabolism in these important medicinal plants. A suite of biotechnological resources, including genetic transformation protocols, has allowed the application of metabolic engineering to modify the alkaloid content of these and related species. An overview of recent progress on benzylisoquinoline and monoterpenoid indole alkaloid biosynthesis in opium poppy and C. roseus is presented.

Quantitative 1H NMR metabolomics reveals extensive metabolic reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures

BackgroundOpium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a model system to study plant alkaloid metabolism. The plant is cultivated as the only commercial source of the narcotic analgesics morphine and codeine, but also produces many other alkaloids including the antimicrobial agent sanguinarine. Modulations in plant secondary metabolism as a result of environmental perturbations are often associated with the altered regulation of other metabolic pathways. As a key component of our functional genomics platform for opium poppy we have used proton nuclear magnetic resonance (1H NMR) metabolomics to investigate the interplay between primary and secondary metabolism in cultured opium poppy cells treated with a fungal elicitor.ResultsMetabolite fingerprinting and compound-specific profiling showed the extensive reprogramming of primary metabolic pathways in association with the induction of alkaloid biosynthesis in response to elicitor treatment. Using Chenomx NMR Suite v. 4.6, a software package capable of identifying and quantifying individual compounds based on their respective signature spectra, the levels of 42 diverse metabolites were monitored over a 100-hour time course in control and elicitor-treated opium poppy cell cultures. Overall, detectable and dynamic changes in the metabolome of elicitor-treated cells, especially in cellular pools of carbohydrates, organic acids and non-protein amino acids were detected within 5 hours after elicitor treatment. The metabolome of control cultures also showed substantial modulations 80 hours after the start of the time course, particularly in the levels of amino acids and phospholipid pathway intermediates. Specific flux modulations were detected throughout primary metabolism, including glycolysis, the tricarboxylic acid cycle, nitrogen assimilation, phospholipid/fatty acid synthesis and the shikimate pathway, all of which generate secondary metabolic precursors.ConclusionThe response of cell cultures to elicitor treatment involves the extensive reprogramming of primary and secondary metabolism, and associated cofactor biosynthetic pathways. A high-resolution map of the extensive reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures is provided.

Opium poppy: blueprint for an alkaloid factory

Opium poppy (Papaver somniferum) produces a large number of benzylisoquinoline alkaloids, including the narcotic analgesics morphine and codeine, and has emerged as one of the most versatile model systems to study alkaloid metabolism in plants. As summarized in this review, we have taken a holistic strategy—involving biochemical, cellular, molecular genetic, genomic, and metabolomic approaches—to draft a blueprint of the fundamental biological platforms required for an opium poppy cell to function as an alkaloid factory. The capacity to synthesize and store alkaloids requires the cooperation of three phloem cell types—companion cells, sieve elements, and laticifers—in the plant, but also occurs in dedifferentiated cell cultures. We have assembled an opium poppy expressed sequence tag (EST) database based on the attempted sequencing of more than 30,000 cDNAs from elicitor-treated cell culture, stem, and root libraries. Approximately 23,000 of the elicitor-induced cell culture and stem ESTs are represented on a DNA microarray, which has been used to examine changes in transcript profile in cultured cells in response to elicitor treatment, and in plants with different alkaloid profiles. Fourier transform-ion cyclotron resonance mass spectrometry and proton nuclear magnetic resonance mass spectroscopy are being used to detect corresponding differences in metabolite profiles. Several new genes involved in the biosynthesis and regulation of alkaloid pathways in opium poppy have been identified using genomic tools. A biological blueprint for alkaloid production coupled with the emergence of reliable transformation protocols has created an unprecedented opportunity to alter the chemical profile of the world’s most valuable medicinal plant.

Inaugural Article: Metabolic engineering of plant alkaloid biosynthesis

Plant alkaloids, one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the biosynthetic pathways for scopolamine, nicotine, and berberine have been cloned, making the metabolic engineering of these alkaloids possible. Expression of two branching-point enzymes was engineered: putrescine N-methyltransferase (PMT) in transgenic plants of Atropa belladonna and Nicotiana sylvestris and (S)-scoulerine 9-O-methyltransferase (SMT) in cultured cells of Coptis japonica and Eschscholzia californica. Overexpression of PMT increased the nicotine content in N. sylvestris, whereas suppression of endogenous PMT activity severely decreased the nicotine content and induced abnormal morphologies. Ectopic expression of SMT caused the accumulation of benzylisoquinoline alkaloids in E. californica. The prospects and limitations of engineering plant alkaloid metabolism are discussed.

Metabolic engineering of plant alkaloid biosynthesis.

Plant alkaloids, one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the biosynthetic pathways for scopolamine, nicotine, and berberine have been cloned, making the metabolic engineering of these alkaloids possible. Expression of two branching-point enzymes was engineered: putrescine N-methyltransferase (PMT) in transgenic plants of Atropa belladonna and Nicotiana sylvestris and (S)-scoulerine 9-O-methyltransferase (SMT) in cultured cells of Coptis japonica and Eschscholzia californica. Overexpression of PMT increased the nicotine content in N. sylvestris, whereas suppression of endogenous PMT activity severely decreased the nicotine content and induced abnormal morphologies. Ectopic expression of SMT caused the accumulation of benzylisoquinoline alkaloids in E. californica. The prospects and limitations of engineering plant alkaloid metabolism are discussed.

Crystallization and preliminary crystallographic study of tropinone reductase II from Datura stramonium.

Tropinone reductase II is an NADPH-dependent oxidoreductase involved in a plant alkaloid metabolism. The enzyme from Datura stramonium has been crystallized using 2-methyl-2,4-pentanediol as a precipitant and a macro-seeding technique. The crystal belongs to space group P42212, with cell dimensions a = b = 62.8 and c = 128.4 A. A data set to 2.6 A resolution has been collected at a cryogenic temperature.

Effect of Nitrogen Nutrition on Alkaloid Metabolism in Plants

It is possible to produce 2–10 fold increases of alkaloids when the plants have been treated with high levels of nitrogen fertilization. The exceptions to this finding were the indole alkaloids and the Solanum glycoalkaloids. We propose a hypothesis to explain how the alkaloid biosynthesis may occur from amino acids in a nitrogen rich and a nitrogen poor plant and this also explains why and how that certain amino acids can be prevented from becoming toxic to the plant by synthesizing a secondary metabolite such as an alkaloid.