Quinolizidine Alkaloid Biosynthesis Research Articles

Biochemical and partial molecular characterization of bitter and sweet forms of Lupinus angustifolius, an experimental model for study of molecular regulation of quinolizidine alkaloid biosynthesis.

The bitter and sweet forms of a plant species differing with alkaloid contents may provide a model system for investigation of alkaloid biosynthesis at a molecular level. The pattern and concentration of quinolizidine alkaloids were determined by capillary GC-MS in bitter and sweet plants of Lupinus angustifolius. Bitter plant contained lupanine, 13alpha-hydroxylupanine, angustifoline, alpha-isolupanine, tetrahydrorhombifoline, and ester-derivatives of 13alpha-hydroxylupanine. In contrast, no alkaloid was detected in sweet plant. The enzymatic activity of acyltransferase for formation of 13alpha-tigloyloxylupanine was similar or even higher in the cell-free extracts of sweet plant than that in bitter plant. These results suggest that the biosynthetic step(s) of ring closure forming the initial cyclic alkaloid, lupanine, from cadaverine is presumably blocked in sweet plant, and that the later steps for modification of the cyclized alkaloids are not altered. We hypothesized that the gene(s) encoding enzyme(s) for ring-closure step might be repressed in sweet plant, and that the expression might take place only in bitter plant. To isolate the genes specifically expressed in bitter plant, cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis was carried out. However, no bitter-specific gene was isolated, suggesting that alkaloid biosynthesis in sweet plant may be down-regulated at a post-transcriptional level.

Subcellular localization of acyltransferases for quinolizidine alkaloid biosynthesis in Lupinus

The subcellular localization of the two acyltransferases, tigloyl-CoA: (−)-13α-hydroxymultiflorine/(+)-13α-hydroxylupanine O-tigloyltransferase (HMT/HLTase) and p-coumaroyl-CoA: (+)-epilupinine O- p-coumaroyltransferase (ECTase), for quinolizidine alkaloid biosynthesis has been investigated using the isolated organelles from the hypocotyls of Lupinus albus and L. hirsutus green seedlings. The two acyltransferases were not located in chloroplasts, where de novo synthesis of the quinolizidine alkaloid skeleton is supposed to take place. The HMT/HLTase activity was associated with the mitochondrial fractions and localized in the matrix, judged by the marker enzyme assay. However, the ECTase activity was not associated with the mitochondrial fractions. Intracellular transport of quinolizidine alkaloids is discussed in relation to the biosynthetic pathway of ester alkaloids in Lupinus plants.

キノリチジンアルカロイドの生合成 生化学的および分子生物学的アプローチ

キノリチジンアルカロイドの生合成 生化学的および分子生物学的アプローチ

Biosynthesis of quinolizidine alkaloids in lupine species

Tracer studies using radiolabelled precursors showed that there was possible interconversion of lupanine and α-isolupanine in Lupinus texensis. Anagyrine and Δ 5-dehydrolupanine are biosynthesized from lupanine via a series of oxidations in L. subcarnosus.

On the Role of Δ1-Piperideine and Tripiperideine in the Biosynthesis of Quinolizidine Alkaloids

Carbon 14-labelled cadaverine, Δ1-piperideine and α-tripiperideine were administered to leaf disks of Lupinuspolyphyllus in short term experiments of 12-20 h duration. Whereas cadaverine was readily incorporated into lupanine in good yields, a 7 to 60 times lower incorporation was observed for tripiperideine or Δ1-piperideine. In L. arboreus the incorporation of cadaverine into sparteine was 9 times better than that of Δ1-piperideine. These results indicate that Δ1-piperideine and tripiperideine are not direct intermediates of lupanine or of sparteine biosynthesis.

Application of 2H n.m.r. spectroscopy to study the incorporation of enantiomeric 2H-labelled cadaverines into quinolizidine alkaloids

The biosynthesis of a number of quinolizidine alkaloids has been studied in two Lupinus species using cadaverine precursors chirally labelled with deuterium. The labelling patterns in lupinine (2), sparteine (3), lupine (6), and angustifoline (8), derived biosynthetically from (R)-[1-2H]-(11) and (S)-[1-2H]cadaverine (12) have been established by 2H n.m.r. spectroscopy. The stereochemistry of a number of the steps involved in the conversion of cadaverine into lupinine (2) and the tetracyclic quinolizidine alkaloids has been established. The presence of 2H at C-17 in samples of sparteine (3), lupanine (6), and angustifoline (8) derived from the (R)-isomer (11) demonstrates that 17-oxosparteine (9) cannot be an intermediate in the biosynthesis of tetracyclic quinolizidine alkaloids. The presence of 2H at C-2 in both samples of sparteine (3) after feeding the precursors (11) and (12) disproves the theory that lupanine (6) is a precursor of sparteine (3).

Quinolizidine alkaloid biosynthesis: incorporation of [1-amino-15N, 1-13C]cadaverine into lupinine

Quinolizidine alkaloid biosynthesis: incorporation of [1-amino-15N, 1-13C]cadaverine into lupinine

Incorporation of chiral [1-2H]cadaverines into the quinolizidine alkaloids sparteine, lupanine, and angustifoline

2 H N.m.r. Spectroscopy has been used to establish the labelling patterns in sparteine (3), lupanine (5), and angustifoline (7), derived biosynthetically from (R)-[1-2H]-(8) and (S)[1-2H]-cadaverine (9); the presence of 2H at C-17 in these three alkaloids from the former precursor (8) demonstrates that 17-oxosparteine cannot be an intermediate in quinolizidine alkaloid biosynthesis.

Evidence for a wide-spread occurrence of the genes of quinolizidine alkaloid biosynthesis: Induction of alkaloid accumulation in cell suspension cultures of alkaloid-‘free’ species

A short-time increase of quinolizidine alkaloid accumulation can be induced in cell suspension cultures of Lupinus polyphyllus 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 alkaloid accumulation in cell cultures of 6 species ( Conium maculatum, Daucus carota, Atropa belladonna, Chenopodium rubrum, Spinacia oleracea, Symphytum officinale), which are supposed to produce other alkaloids or no alkaloids at all. This indicates that the genes of quinolizidine alkaloid biosynthesis are obviously not restricted to the Fabaceae family but are widely distributed in higher plants.

Accumulation of Quinolizidine Alkaloids in Plants and Cell Suspension Cultures: Genera Lupinus, Cytisus, Baptisia, Genista, Laburnum, and Sophora

The patterns of quinolizidine alkaloids in cell cultures of 10 species of Fabaceae were analyzed by high-resolution GLC and GLC-MS and compared with the alkaloids present in the leaves of the respective plants. Lupanine was produced in all 10 cell suspension cultures as the main alkaloid. It was accompanied by sparteine, tetrahydrorhombifoline, 17-oxosparteine, 13-hydroxylupanine, 4-hydroxylupanine, 17-oxolupanine, and 13-hydroxylupanine esters as minor alkaloids in some species. The alkaloid patterns of the plants differed markedly in that alpha-pyridone alkaloids were the major alkaloids in the genera Cytisus, Genista, Laburnum and Sophora but were not accumulated in the cell cultures. These data further support the assumption that the pathway leading to lupanine is the basic pathway of quinolizidine alkaloids biosynthesis and that the other alkaloids are derived from lupanine.

Lysine decarboxylase in plants and its integration in quinolizidine alkaloid biosynthesis

In leaves of alkaloid producing Lupinus polyphyllus lysine decarboxylase activity is positively correlated with the chlorophyll content during leaf regreening. A similar positive correlation was found between the leaf alkaloid content and lysine decarboxylase activity in L. albus and L. luteus. These results indicate that in lupin leaves lysine decarboxylase is an integrated part of the alkaloid specific biosynthetic sequence. Lysine decarboxylase could also be demonstrated in 46 alkaloidal and non-alkaloidal species out of 17 families of higher plants, including cell cultures of seven species.

Quinolizidine alkaloid biosynthesis: incorporation of [1-amino-15N,1-13C]cadaverine into sparteine

Three [1-amino-15N,1-13C]cadaverine (4) units are incorporated to about the same extent into sparteine (5); the presence of only two 13C–15N doublets in the 13C{1H} n.m.r. spectrum of sparteine indicates that two of these units are transformed into the outer rings of sparteine in a specific fashion.

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

Studies with purified chloroplasts of Lupinus polyphyllus 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.

Diurnal Fluctuation of Quinolizidine Alkaloid Accumulation in Legume Plants and Photomixotrophic Cell Suspension Cultures

Abstract Fluctuations of quinolizidine alkaloid content in leaflets of Lupinus polyphyllus, L. hartwegii, Baptisia australis, and Sarothamnus scoparius were studied over a 36 h period. The alkaloid contents reached their maximum at noon or early afternoon, and their minimum during the night. The amplitudes of diurnal variations in alkaloid levels lay within a range of 60 to 470% of the early morning contents of alkaloids (= 100%) in leaves. In photomixotrophic cell suspension cultures of L. polyphyllus and S. scoparius cultured under day-night regime, a similar increase of the alkaloid levels was observed within 5 to 8 h after onset of illumination. A subsequent excretion of the alkaloids produced, which also followed a diurnal rhythm, was found. S. scoparius cells excreted lupanine, L. polyphyllus cells 13-cinnamoyloxylupanine. From the cell culture and plant experiments it can be assumed that the alkaloids which are formed during illumination are translocated to some degree afterwards. In cell suspension cultures the alkaloids are subjected to rapid and rhythmic turnover. Exogenous alkaloids, fed to the cultures, are taken up by the cells and disappear usually within the first 72 h. A possible mechanism for a light-mediated regulation of quinolizidine alkaloid biosynthesis which was found to be localized in leaf chloroplast, is discussed.

Quinolizidine Alkaloid Composition of Plants and of Photomixotrophic Cell Suspension Cultures ofSarothamnus scopariusandOrobanche rapum-genistae

The alkaloid composition of Sarothamnus scoparius cell cultures, plants and of its root parasite Orobanche rapum-genistae were analyzed by capillary gas-liquid chromatography (GLC) and capillary GLC/mass spectrometry. S. scoparius plants were found to contain the already known isosparteine, sparteine, 17-oxosparteine, lupanine and other quinolizidine alkaloids such as ammodendrine, N-methylangustifoline, alpha-isolupanine, 5,6-dehydrolupanine, 4-hydroxylupanine, 17-oxolupanine and five 13-hydroxylupanine esters. Two new alkaloids could be identified, a dehydrosparteine (presumably the 11,12-dehydroderivative) and a dihydroxylupanine (presumably 4,13-dihydroxylupanine). The distribution of these alkaloids within the different plant organs is reported. Orobanche rapum-genistae plants contain the already known sparteine, lupanine and 13-hydroxylupanine and 12 other lupin alkaloids, which are present in the Sarothamnus host plant. Photomixotrophic cell suspension cultures of S. scoparius accumulate lupanine as the main alkaloid. On tissue differentiation (bud formation) the cultures accumulate sparteine as well as lupanine. Whereas sparteine is stored in the cells, lupanine is excreted into the medium. Alkaloid formation of the cell cultures is positively correlated with chlorophyll content of the cells. The alkaloid pattern found in S. scoparius supports the hypothesis that lupanine biosynthesis is the common route of quinolizidine alkaloid biosynthesis from which the other alkaloids such as sparteine are derived.

Activation of chloroplast-localized enzymes of quinolizidine alkaloid biosynthesis by reduced thioredoxin

Previous studies from the laboratory of the authors have shown that the tetracyclic quinolizidine alkaloids are synthesized in leaf chloroplasts of Lupinus polyphyllus. Additionally, alkaloid formation reveals a light dependent diurnal rhythm in vivo. The present study shows that the principal biosynthetic enzymes, lysine decarboxylase and 17-oxosparteine synthase, assayed in acetone powder extracts and isolated chloroplasts of L. polyphyllus, were activated by reduced E. coli thioredoxin. Since both enzymes display optimal activity at pH 8 and were rather inactive at pH 7, both thioredoxin and the light mediated shift in the hydrogen ion concentration of the chloroplast stroma from pH 7 to pH 8 may be involved in the light controlled alkaloid formation.

Sites of Enzymatic Synthesis of Quinolizidine Alkaloids and Their Accumulation in Lupinus polyphyllus

Summary The distribution of the first two enzymes of the biosynthetic pathway of quinolizidine alkaloid biosynthesis, lysine decarboxylase and 17-oxosparteine synthase, was studied in the various organs of a flowering Lupinus polyphyllus plant. Lysine decarboxylase activity is present in all plant parts, whereas the activiy of the key enzyme 17-oxosparteine synthase is only expressed in leaf extracts. This indicates that alkaloid synthesis is bound to the leaves, where both enzyme had been localized in chloroplasts previously. The alkaloid content, alkaloid concentration and the alkaloid composition was quantitatively evaluated for the various organs of two flowering L. polyphyllus plants. The highest alkaloid content was found in the roots (38-45% of total alkaloids) and leaves (27-31%), but alkaloids are virtually present in all parts of a plant. Relatively high alkaloid concentrations were found in leaves, in younger parts of the shoot axis and in mature seeds. The alkaloid content of leaves is reduced during senescence by 90% as compared with young leaves. The alkaloid pattern evaluated quantitatively by capillary gas chromatography for the organs of two plants and seeds includes the following compounds: lupanine, sparteine, angustifoline, N-methylangustifoline, dehydrolupanine, 17-oxolupanine, 13-hydroxylupanine and its esters (13-tigloyl-, 13-angeloyl-, 13-benzoyl- and 13-cis and 13-transcinnamoyl-oxylupanine). A high percentage of ester alkaloids and a low content of 13-hydroxylupanine was found in leaves. Roots and seeds, the main sites of alkaloid accumulation, displayed the opposite pattern, i.e. high concentration of 13-hydroxylupanine and low concentration of ester alkaloids.

Light-Mediated Control of Quinolizidine Alkaloid Biosynthesis

Light-Mediated Control of Quinolizidine Alkaloid Biosynthesis

Enzymatic Synthesis of Quinolizidine Alkaloids in Lupin Chloroplasts

Abstract The enzymatic sequence responsible for the biosynthesis of tetracyclic quinolizidine alkaloids could be localized in chloroplasts isolated from Lupinus polyphyllus leaves and L. albus seedlings by differential centrifugation. Upon feeding of cadaverine to isolated chloroplasts lupanine is produced as the main alkaloid. Chloroplasts treated with digitonine produce sparteine and 17-oxosparteine instead of lupanine, thus indicating that the biosynthetic sequence is interrupted. The intermediacy of 17-oxosparteine could be confirmed since exogeneous 17-oxosparteine is converted into lupanine by intact chloroplasts. 17-Oxosparteine synthase (see Z. Naturforsch. 34 c, 704 1979) the key enzyme of quinolizidine alkaloid biosynthesis could be solubilized from chloro­ plasts treated with detergents or osmotic shock.

Cadaverine-pyruvate transamination: The principal step of enzymatic quinolizidine alkaloid biosynthesis in Lupinus polyphyllus cell suspension cultures

Cadaverine-pyruvate transamination: The principal step of enzymatic quinolizidine alkaloid biosynthesis in Lupinus polyphyllus cell suspension cultures