Alkaloid Pattern of Cell Suspension Cultures and Differentiated Plants of Lupinus polyphyllus

Wink, Michael; Witte, Ludger; Schiebel, H. M.; Hartmann, Thomas

doi:10.1055/s-2008-1074868

Cited by 61 publications

(50 citation statements)

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“…Upon application of the com~unds which induce alkaloid accumulation in lupins (table 1) we could observe a significant increase in quinolizidine alkaloids 2-72 h after the treatment (table 2). These alkaloids could be unambiguously identified by GLC/MS and authentic reference compounds [5,6,10] as lupanine, the major alkaloid, along with sparteine, tetrahydrorhombifoline, and 17-oxosparteine as minor alkaloids. The activity of oxosparteine synthease was not significantly influenced by the inducers.…”

Section: Induction Of Quinolizidine Alkaloid Accumulation In Cell Culmentioning

confidence: 96%

Evidence for a wide‐spread occurrence of the genes of quinolizidine alkaloid biosynthesis

Wink

Witte

1983

FEBS Letters

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A short-time increase of quinolizidine alkaloid accumulation can be induced in cell suspension cultures of Lupinus~lyphyll~ by application of foreign alkaloids such as papaverine, coniine, and other compounds like CAMP, polyamines, or even by transfer of the cells into fresh, autoclaved cell culture medium. This induction can be inhibited by cycloheximide. Using the same method of induction we were able to show quinolizidine afkafoid accumulation in ceil cultures of 6 species (Conium macufafum, Duucus carofa, Atropa belladonna, Chenopodium rubrum, Spinacia oieracea, Symphytum officinaie), which are supposed to produce other alkaloids or no alkaloids at all. This indicates that the genes of quinoliiidine alkaloid biosynthesis are obviously not restricted to the Fabaceae family but are widely distributed in higher plants. fnduetionCell ~spension culture Gene, of alkaloid biosynthesis A lkaioid-ffree ' species 1. I~RO~UCTION Plant natural products, such as flavonoids and lignins, are present in most species, whereas other secondary compounds (e.g., alkaloids) are thought to be present in a limited number of species, genera, or families. Quino~zidine alkaloids are widely distributed in the Fabaceae and are assumed to be specific for this plant family [ 11. Stimulated by experiments [2,3], which implied a wider distribution of lupin alkaloids, we studied if quinolizidine alkaloid biosynthesis takes place also in families other than the Fabaceae. As an experimental approach, we tried modulating the alkaloid metabolism of plant cell suspension cultures of alkaloid-producing species and of alkaloid-'free' species. MATERIALS AND METHODSCell suspensions of Lupinus polyphyllus, L. luteus, Conium maculatum, Daucus carota, Atropa belladonna, Chenopodium rubrum, Spinacea oleracea, and Symphytum of~~inale were cultured at 25"C, and 16 h of daily illumination (3000 lux) as in [3-61.The activity of oxosparteine synthase [7,8] was assayed according to [9], 2.1. A fkafoid extraction Cells were homogenized in 0.5 M HCl and left standing at room temperature for 30 min. After filtering the homogenate through nylon gauze (100 pm mesh) the filtrate was alkalinized with 25% ammonium hydroxide and applied onto a standard 196Pubbsited by Ekevier Science Publishers B. V.

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Section: Induction Of Quinolizidine Alkaloid Accumulation In Cell Culmentioning

confidence: 96%

Evidence for a wide‐spread occurrence of the genes of quinolizidine alkaloid biosynthesis

Wink

Witte

1983

FEBS Letters

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“…The Kovats index was determined by co-chromatography of a mixture of linear alkanes. Quinolizidine alkaloids were identified according to their Kovats retention indices (RI) (Wink et al 1981, molecular ion (M + ) and the significant fragments and their relative abundance (Wink et al 1980;Wink et al 1981Wink et al , 1982Wink et al , 1983. Sparteine (100 lg ml -1 ) was used as external standard.…”

Section: Characterization Of Alkaloidsmentioning

confidence: 99%

Activity of quinolizidine alkaloids from three Mexican Lupinus against the lepidopteran crop pest Spodoptera frugiperda

et al. 2008

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Bitter lupins (Lupinus spp.) are not used as a protein source because of their toxicity. However, they may have alternative uses as potential sources of natural insecticides. Quinolizidine alkaloids (QA) of three Mexican Lupinus species (Fabaceae): L. montanus (HBK), L. stipulatus (Agardh) and L. aschenbornii (Schauer), were analyzed by capillary Gas Chromatography-Mass Spectrometry. Sparteine was found in high amounts in both L. montanus and L. aschenbornii while the major alkaloids in L. stipulatus extract were aphylline and an epiaphylline-like compound. Alkaloid extracts were tested for their insecticidal activity using larvae of the Fall Armyworm, Spodoptera frugiperda (Smith); (Lepidoptera, Noctuidae) as a model pest. We compared LD 50 values and mean weight of caterpillars fed with alkaloid extracts of the three species studied with those of sparteine, a widespread QA found in various lupin species. Extracts of L. montanus and L. aschenbornii were found to be as effective as sparteine and extracts L. stipulatus were found to be the most toxic against the larvae of S. frugiperda. This suggests that the various QA act differently on caterpillars, and could be used to control Spodoptera populations.

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“…by HPLC. The identity of all alkaloids has been confirmed by capillary GLC and GLC-MS (20,21 Gradient-Centrifugation. About 2 ml of the above chloroplast solution was layered onto a discontinuous sucrose gradient (5 ml 57% w/v sucrose, 6 ml 50% sucrose, 6 ml 43% sucrose, 6 ml 33% sucrose, and 8 Isolation of Chloroplast Stroma and Thylakoids.…”

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confidence: 93%

Localization of the Enzymes of Quinolizidine Alkaloid Biosynthesis in Leaf Chloroplasts of Lupinus polyphyllus

Hartmann¹

1982

Plant Physiol.

111

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Studies with purified chioroplasts of Lupinuspolyphyllw LINDL. leaflets indicate that the first two enzymes of quinolizidine alkaloid biosynthesis, lysine decarboxylase and 17-oxosparteine synthase, are localized in the chloroplast stroma. Thus, both enzymes share the same subcellular compartment as the biosynthetic pathway of lysine, the precursor of quinolizidine alkaloids. The activity of diaminopimelate decarboxylase, the final enzyme in lysine biosynthesis, is about two to three orders of magnitude higher than that of the enzymes of alkaloid formation.Tetracyclic quinolizidine alkaloids such as lupanine are derived from lysine via the symmetrical intermediate cadaverine ( Fig. 1 About 30 ml of the homogenate, pipetted into 40-ml centrifuge tubes, was underlayered with 10 ml Percoll solution according to Mills and Joy (6) (40% v/v Percoll, 330 mM D-mannitol, 50 mm Tricine, pH 7.9, 0.1% w/v BSA). Intact chloroplasts were pelleted by centrifugation at 2,500g for 2 min in a RC-5B centrifuge (Sorvall) equipped with the fixed-angle rotor SS 34. The supernatant and then the Percoll layer were removed by aspiration. The chloroplast pellet was resuspended in the following buffer solution by repeated pipetting: 330 mM D-mannitol, 5 mM ascorbate, 2 mM EDTA, 20 mm NaCl, 1 mM MgCl2, 1 mrm MnCl2, 2 mm NaNO3, 50 mM Hepes (pH 7.6). This fraction was designated as the fraction containing 'intact chloroplasts' (6).Gradient-Centrifugation. About 2 ml of the above chloroplast solution was layered onto a discontinuous sucrose gradient (5 ml 57% w/v sucrose, 6 ml 50% sucrose, 6 ml 43% sucrose, 6 ml 33% sucrose, and 8 ml 20% sucrose in 50 mm Tricine [pH 8] each). Centrifugation in a SS 90 vertical rotor was performed at 18,000 rpm for 30 min in a Sorvall RC-5B centrifuge equipped with a special rate controller. An Isco model UA 5 fractionator was used to fractionate the gradients. Fractions of 2 ml were collected.Isolation of Chloroplast Stroma and Thylakoids. Intact chloroplasts were isolated by the Percoll method. The chloroplast pellet was suspended in 35 ml hypotonic buffer (10 mm Tricine, pH 7.6, 4 mM MgCI2) and left standing in an ice-bath for 10 min. The chloroplasts were further disrupted with a Potter homogenator (one stroke), and 9 ml were layered onto a discontinuous sucrose gradient, consisting of 4 ml 1.5 M sucrose, 5 ml 1.2 M sucrose, 7 ml 0.93 M sucrose, and 9 ml 0.6 M sucrose in 10 mm Tricine (pH 7.6) and 4 mm MgCl2. The gradients were centrifuged in the SS 90 vertical rotor for 20 mim at 15,000 rpm. We assume that the upper fractions of the gradient, which contain tne soluble portion of the chloroplasts, represent the stroma, whereas the lower dark green band contains mainly the thylakoids (3). We were not able to recover a fraction containing the chloroplast envelopes. Stromal and thylakoid fractions were removed by aspiration. The thylakoid fraction was suspended in 15 ml 0.1 M phosphate buffer (pH 8) and centrifuged for 10 min at 10,000g. The resulting pellet was resuspended in 8 ml phosphate buffer...

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Alkaloid Pattern of Cell Suspension Cultures and Differentiated Plants of Lupinus polyphyllus

Cited by 61 publications

References 1 publication

Evidence for a wide‐spread occurrence of the genes of quinolizidine alkaloid biosynthesis

Evidence for a wide‐spread occurrence of the genes of quinolizidine alkaloid biosynthesis

Activity of quinolizidine alkaloids from three Mexican Lupinus against the lepidopteran crop pest Spodoptera frugiperda

Localization of the Enzymes of Quinolizidine Alkaloid Biosynthesis in Leaf Chloroplasts of Lupinus polyphyllus

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