Metabolic links between the biosynthesis of pyrrolizidine alkaloids and polyamines in root cultures of Senecio vulgaris

Isotope feeding and inhibitor experiments were performed in order to elucidate the pathway common to polyamine and alkaloid biosynthesis in root cultures of Senecio vulgaris L. -Difluoromethylarginine, a specific inhibitor of arginine decarboxylase, prevented completely the incorporation of radioactivity from [¹⁴C]arginine and [¹⁴C]ornithine into spermidine and the pyrrolizidine alkaloid senecionine N-oxide. In contrast, -difluoromethylornithine, a specific ornithine-decarboxylase inhibitor, had no effect on the flow of radioactivity from labelled ornithine and arginine into polyamines and alkaloids. Thus, putrescine, the common precursor of polyamines and pyrrolizidine alkaloids, is exclusively derived via the arginine-agmatine route. Ornithine is rapidly transformed into arginine. Recycling of the guanido moiety of agmatine back to ornithine can be excluded. Putrescine and spermidine were found to be reversibly interconvertable and to excist in a highly dynamic state. In contrast, senecionine N-oxide did not show any turnover but accumulated as a stable metabolic product. In-vivo evidence is presented that the carbon flow from arginine into the polyamine/alkaloid pathway may be controlled by spermidine. The possible importance of the metabolic coupling of pyrrolizidine-alkaloid biosynthesis to polyamine metabolism is discussed.Abbreviations DFMA D,l--difluoromethylarginine - DFMO D,l--difluoromethylornithine - FW fresh weight     

Sites of synthesis,translocation and accumulation of pyrrolizidine alkaloid N-oxides in Senecio vulgaris L.

¹⁴C-Labelled alkaloid precursors (arginine, putrescine, spermidine) fed to Senecio vulgaris plants via the root system were rapidly taken up and efficiently incorporated into the pyrrolizidine alkaloid senecionine N-oxide (sen-Nox) with total incorporations of 3–6%. Considerable amounts of labelled sen-Nox were translocated into the shoot and were directed mainly into the inflorescences, the major sites of pyrrolizidine-alkaloid accumulation. Detached shoots of S. vulgaris were unable to synthesize pyrrolizidine alkaloids, indicating that the roots are the site of their biosynthesis. Further evidence was obtained from studies with in-vitro systems established from S. vulgaris: root cultures were found to synthesize pyrrolizidine alkaloids but not cell-suspension cultures, tumor cultures or shoot-like teratomas obtained by transformation with Agrobacterium tumefaciens. Studies on transport of [¹⁴C]sen-Nox, which was fed either to detached shoots or to the root system of intact plants, indicate that the alkaloid N-oxide does not simply follow the transpiration stream but is specifically channelled to the target tissues such as epidermal stem tissue and flower heads. Exogenously applied [¹⁴C]senecionine is rapidly N-oxidized. If the phloem path along the stem is blocked by a steam girdle translocation of labelled sen-Nox is blocked as well. Root-derived sen-Nox accumulated below the girdle and only trace amounts were found in the tissues above. It is most likely that the root-to-shoot transport of sen-Nox occurs mainly if not exclusively via the phloem. In accordance with previous studies the polar, salt-like N-oxides, which are often considered to be artifacts, were found to be the real products of pyrrolizidine-alkaloid biosynthesis as well as the physiological forms for long-distance transport, tissue-specific distribution and cellular accumulation.Abbreviations FW fresh weight - sen senecionine - sen-Nox senecionine N-oxide     

Chemical diversity and variation of pyrrolizidine alkaloids of the senecionine type: biological need or coincidence?

Laboratory and field tracer experiments with ¹⁴C-labelled senecionine N–oxide (SO) and distant biosynthetic precursors such as [¹⁴C]putrescine revealed that pyrrolizidine alkaloid N-oxides (PAs) in Senecio vernalis Waldstr. & Kit. (Asteraceae) show no significant turnover over periods of up to 29 d. However, PAs are spatially mobile, they are continuously allocated, and labelled PAs are even detectable in leaves and capitula developed weeks after tracer application. Chemical diversification of SO, the common product of PA biosynthesis in roots, was studied in five Senecio species (i.e. S. vernalis Waldstr. & Kit., S. vulgaris L, S. inaequidens DC, two chemotypes of S. jacobaea L. and S. erucifolius L.). Tracer experiments revealed that shoots are capable of transforming [¹⁴ C]SO into the unique species–specific PA patterns. Within a plant, the transformation efficiency of SO can vary quantitatively and qualitatively between shoot organs (i.e. leaves, stems and inflorescences). All transformations proceed position-specifically and stereoselectively. They comprise simple one-step or two-step reactions such as hydroxylations, epoxidations, dehydrogenations, and O-acetylations, as well as the more complex conversion of the retronecine into the otonecine base moiety (e.g. SO into senkirkine). Taking all the evidence together, the qualitative and quantitative composition of the Senecio PA pattern is a dynamic and sensitive equilibrium between a number of interacting processes: (i) constant rate of de-novo synthesis of SO in roots, (ii) continuous long-distance translocation of SO into shoots, (iii) efficiency of SO transformations which may vary between plant organs, (iv) continuous allocation of PAs in the plant, and (v) efficiency and tissue selectivity of vacuolar storage. We suggest that in constitutive plant defence, without significant turnover of its components, such a highly plastic system provides a powerful strategy to successfully defend and possibly escape herbivory. Received: 27 March 1998 / Accepted: 19 May 1998     

Alkaloid N-oxides as transport and vacuolar storage compounds of pyrrolizidine alkaloids in Senecio vulgaris L

Cell-suspension cultures of pyrrolizidinealkaloid-producing species selectively take up and accumulate senecionine (sen) and its N-oxide (sen-Nox). Cultures established from non-alkaloid-producing species are unable to accumulate the alkaloids. The uptake and accumulation of ¹⁴C-labelled alkaloids was studied using a Senecio vulgaris cell-suspension culture as well as protoplasts and vacuoles derived from it. The alkaloid uptake exhibits all characteristics of a carrier-mediated transport. The uptake of sen-Nox follows a multiphasic saturation kinetics. The K_m-values for sen Nox of 53 M and 310 M are evaluated. Senecionine competitively inhibits sen-Nox uptake, indicating that the tertiary alkaloid and its N-oxide share the same membrane carrier. The N-oxide of sen shows a pH optimum below 5.5, whereas sen is taken up over a range from pH 4 to 8. Activation energies of 90 and 53 kJ·mol^-1 are calculated for sen-Nox and sen transport, respectively. At concentrations of 10 to 100 M, sen-Nox is rapidly taken up by cells and protoplasts; within 2 h >90% of total N-oxide is within the cells. By contrast the uptake of sen is less efficient. Vacuoles isolated from protoplasts preloaded with sen-Nox totally retained the alkaloid N-oxide, whereas sen is rapidly lost during the procedure of vacuole preparation. N-oxidation converts the weak lipophilic tertiary base into a charged polar molecule which is excellently adapted to serve as the cellular transport and storage form of pyrrolizidine alkaloids.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - DNP 2,4-dinitrophenol - sen senecionine - sen-Nox senecionine N-oxide     

Alkaloid patterns and biosynthetic capacity of root cultures from some pyrrolizidine alkaloid producing Senecio species

Root cultures of Senecio vulgaris, S. vernalis, S. erucifolius and S. squalidus were established. The patterns of pyrrolizidine alkaloids found in these root cultures were analyzed by high-resolution GC and GC-MS and compared with the alkaloids present in the respective plants. In vitro cultured roots produce alkaloid patterns and accumulate quantities which are comparable to those found in soil grown plants. With the exception of the otonecine derivative senkirkine all pyrrolizidines accumulate as N-oxides. Only senkirkine is partially released into the medium. The cultures incorporate biosynthetic precursors, e.g. ¹⁴C-labelled putrescine or spermidine with high efficiency into the alkaloids. Senecionine N-oxide was found to be the main product of biosynthesis. Evidence is presented that senecionine N-oxide is directly transformed into senkirkine, the main alkaloid of S. vernalis root cultures.Abbreviations GC Gas chromatography - MS Mass spectroscopy - PND Phosphorous-Nitrogen-Detector - FID Flame Ionization Detector - fr.wt Fresh weight     

Studies on the biosynthesis of tropane alkaloids by Datura stramonium L. transformed root cultures

Transformed root cultures of Datura stramonium, competent in tropane-alkaloid biosynthesis, have been treated with exogenous plant growth regulators. It was found that combinations of -naphthalene-acetic acid, kinetin (N⁶-furfurylaminopurine) and 2,4-dichlorophenoxyacetic acid induced de-differentiation, causing both the rooty phenotype and the hyoscyamine-biosynthetic capacity to be lost. Alkaloid biosynthesis disappeared rapidly and prior to the loss of morphological integrity. It was observed that the enzymes ornithine decarboxylase (EC 4.1.1.17), arginine decarboxylase (EC 4.1.1.19) and N-methylputrescine oxidase did not show the increase in level normally associated with subculturing the roots. The level of putrescine N-methyltransferase (EC 2.1.1.53) activity, the first enzyme fully committed to hyoscyamine biosynthesis, rapidly declined, about 80% being lost from the roots within 12h. This activity, although showing some temporary restoration, declined further after a few days, and was totally absent from fully dispersed cultures. N-Methylputrescine oxidase persisted at a low level. Following sub-culture of established de-differentiated lines to plant-growth-regulator-free medium, limited root regeneration occurred. The roots formed showed renewed competence in alkaloid biosynthesis and putrescine N-methyltransferase and N-methylputrescine oxidase activities were restored to their normal levels. The relationship between the morphological state and alkaloid-biosynthetic capacity of the cultures is discussed in relation to the overall control of alkaloid biosynthesis.Abbreviations ADC arginine decarboxylase - FW fresh weight - MPO N-methylputrescine oxidase - NAA -naphthalineacetic acid - ODC ornithine decarboxylase - pgr plant growth regulator - PMT putrescine N-methyltransferase We are most grateful to Abigael Peerless and Bridget Chapman for assistance with various part of this work.     

Polyamines in Rice Seedlings under Oxygen-Deficit Stress

Incubation of 3-d-old seedlings of Oryza sativa L. cv Arborio under anaerobic conditions, leads to a large increase in the titer of free putrescine while aerobic incubation causes a slight decrease. After 2 days, the putrescine level is about 2.5 times greater without oxygen than in air. The rice coleoptile also accumulates a large amount of bound putrescine and, to a lesser extent, spermidine and spermine (mainly as acid-soluble conjugates). Accumulation of conjugates in the roots is severely inhibited by the anaerobic treatment. Feeding experiments with labeled amino acids showed that anoxia stimulates the release of ¹⁴CO₂ from tissues fed with [¹⁴C]arginine and that arginine is the precursor in putrescine biosynthesis. After 2 d of anoxia, the activity of arginine decarboxylase was 42% and 89% greater in coleoptile and root, respectively, than in the aerobic condition. The causes of the differences in polyamine metabolism in anoxic coleoptiles and roots are discussed.     

Products of fermentation in the roots of alders (Alnus Mill.)

The aim of this work was to discover the products of carbohydrate fermentation in alder roots. Experiments were done with roots of trees growing in naturally wet soils. Detached, anaerobic roots accumulated ethanol, and ethanol was the major labelled product of metabolism of [U-¹⁴C]sucrose. Glycerol was not labelled from [U-¹⁴C]sucrose, and did not accumulate in detached or attached roots. In both winter and summer, roots in the field contained little or no glycerol, and the amount was less than that in the aerobic parts of the tree. Roots in the field contained substantial amounts of ethanol. We conclude that ethanol is the major product of fermentation in alder roots, and that glycerol is not a significant product. These results are not consistent with Crawford's metabolic theory of flooding tolerance.     

Polyamines and Root Formation in Mung Bean Hypocotyl Cuttings : II. Incorporation of Precursors into Polyamines

The incorporation of [¹⁴C]arginine and [¹⁴C]ornithine into various polyamines was studied in mung bean (Vigna radiata [L.] Wilczek) hypocotyl cuttings with respect to the effect of indole-3-butyric acid on adventitious root formation.

Both [¹⁴C]arginine and [¹⁴C]ornithine are rapidly incorporated into putrescine, spermidine, and spermine, with similar kinetics, during 5- to 24-hour incubation periods. The incorporation of arginine into putrescine is generally higher than that of ornithine. The biosynthesis of putrescine and spermidine from the precursors, in the hypocotyls, is closely related to the pattern of root formation: a first peak at 0 to 24 hours corresponding to the period of root primordia development, and a second peak of putrescine biosynthesis at 48 to 72 hours corresponding to root growth and elongation. Indole-3-butyric acid considerably enhances putrescine biosynthesis in both phases, resulting in an increase of the putrescine/spermidine ratio.

It is concluded that the promotive effect of indole-3-butyric acid on putrescine biosynthesis, from both arginine and ornithine, supports the hypothesis that auxin-induced root formation may require the promotion of polyamine biosynthesis.

     

Synthesis and Content of Polyamines in Bloodstream Trypanosoma brucei*

SYNOPSIS. The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [³H]ornithine, [¹⁴C]arginine and [¹⁴C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [¹⁴C]methionine. Putrescine and spermidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous ¹⁴C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.     

Reduced gravitropic sensitivity in roots of a starch-deficient mutant ofNicotiana sylvestris

The activity of arginine decarboxylase (EC 4.1.1.19) in cultured roots of Hyoscyamus albus L., which produce considerable amounts of tropane alkaloids, was twice that of ornithine decarboxylase (EC 4.1.1.17), both activities being highest during active root growth, whereas arginase (EC 3.5.3.1) activity was negligible. Actively growing roots had putrescine conjugates as their major polyamines, and spermidine was the most abundant free polyamine. Putrescine N-methyltransferase (PMT; EC 2.1.1.53) activity was high, the peak occurring on the sixth day of culture when root growth became slower. Thereafter, the free N-methylputrescine content of the roots increased and was followed by an increase in alkaloid content (mostly hyoscyamine). The amounts of arginine and, especially, of ornithine were low. No N-methylornithine was detected. The PMT activity was present only in root, shoot and cell-suspension cultures of plants that synthesized tropane alkaloids or nicotine; no enzyme activities that methylate ornithine at the -amino group or that decarboxylate -N-methylornithine were detected in any of the cultures tested. Our data indicate that tropane alkaloids in H. albus roots are synthesized by way of the symmetrical putrescine, i.e. a pathway different from that proposed by E. Leete (1962, J. Am. Chem. Soc. 84, 55) according to which these alkaloids are synthesized by way of asymmetrical -N-methylornithine.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - PCA perchloric acid - PMT putrescine N-methyltransferase     

In vivo nuclear-magnetic-resonance analysis of polyamine and alkaloid metabolism in transformed root cultures of Datura stramonium L.: evidence for the involvement of putrescine in phytohormone-induced de-differentiation

The utilisation and accumulation of ¹⁵N-labeled metabolites by a ¹⁵N-labeled transformed root culture of Daturastramonium L. was investigated by in vivo ¹⁵N-nuclear-magnetic-resonance (NMR) spectroscopy. After resuspension in spent growth medium, the pools of [¹⁵N]glutamate and [¹⁵N]glutamine were rapidly depleted and there was an increase in the ¹⁵N-NMR signals from conjugated putrescines and hyoscyamine. The signal from the conjugated putrescines passed through a maximum 2 d after the roots were resuspended, and it was concluded that putrescine could be stored as putrescine conjugates prior to its utilisation in other pathways. The transient accumulation of ¹⁵N-label in the hydroxy-cinnamoylputrescines was reduced when the de-differentiation of the root cultures into a suspension culture was initiated by exposure to a medium containing α-napthaleneacetic acid and kinetin. This led to the hypothesis that phytohormone-induced de-differentiation of the root cultures required the presence of free polyamines, and this was tested using two potent inhibitors of putrescine biosynthesis, dl-α-difluoromethylarginine and dl-α-difluoromethylornithine. In-vivo ¹⁵N-NMR spectra of roots grown in ¹⁵N-enriched medium supplemented with these inhibitors showed that the ¹⁵N-labelling of the conjugated polyamines and hyoscyamine was markedly reduced. dl-α-difluoromethylarginine also prevented the phytohormone induced de-differentiation of the root cultures, and this effect could be reversed by the supply of exogenous putrescine. Thus the supply of putrescine appears to play a crucial role in mediating the phytohormone induced de-differentiation of the root culture. Received: 13 September 1997 / Accepted: 12 November 1997     

Increased levels of glycerol-3-phosphate lead to a stimulation of flux into triacylglycerol synthesis after supplying glycerol to developing seeds of<Emphasis Type="Italic"> Brassica napus</Emphasis> L.<Emphasis Type="Italic"> in planta</Emphasis>

Glycerol-3-phosphate (glycerol-3P) is a primary substrate for triacylglycerol synthesis. In the present study, changes in the levels of glycerol-3P during rape (Brassica napus L.) seed development and the influence of manipulating glycerol-3P levels on triacylglycerol synthesis were investigated. (i) Glycerol-3P levels were high in young seeds and decreased during seed development at 30 and 40 days after flowering (DAF), when lipid accumulation was maximal. (ii) To manipulate glycerol-3P levels in planta, various concentrations of glycerol were injected directly into 30-DAF seeds, which remained otherwise intact within their siliques and attached to the plant. Injection of 0–10 nmol glycerol led to a progressive increase in seed glycerol-3P levels within 28 h. (iii). Increased levels of glycerol-3P were accompanied by an increase in the flux of injected [¹⁴C]sucrose into total lipids and triacylglycerol, whereas fluxes to organic acids, amino acids, starch, protein and cell walls were not affected. (iv) When [¹⁴C]acetate was injected into seeds, label incorporation into total lipids and triacylglycerol increased progressively with increasing glycerol-3P levels. (v) There was a strong correlation between the level of glycerol-3P and the incorporation of injected [¹⁴C]acetate and [¹⁴C]sucrose into triacylglycerol. (v) The results provide evidence that the prevailing levels of glycerol-3P co-limit triacylglycerol synthesis in developing rape seeds.Abbreviations DAF Days after flowering - DAG Diacylglycerol - G3PAT Glycerol-3-phosphate acyltransferase - Glycerol-3P Glycerol-3-phosphate - PA Phosphatidic acid - PC Phosphatidylcholine - TAG Triacylglycerol,     

Amine biosynthesis in Lathyrus sativus seedlings

The biosynthesis of certain amines in Lathyrus sativus seedlings was studied in isolated shoots and cotyledons. In shoots, arginine was about 14 times more efficient than ornithine for the synthesis of agmatine, putrescine, spermidine and spermine. Isotope dilution experiments, and the changes in specific activities of the 4 amines with time when ¹⁴C-arginine served as the precursor, indicated that putrescine and the polyamines were formed mainly from arginine, via agmatine. Similar experiments showed that cadaverine was formed at least in part from homoarginine, though lysine was ca 4 times more effective as a precursor. The pattern of changes in specific activity of homoagmatine and cadaverine with time when ¹⁴C-homoarginine served as the precursor support the conclusion that homoarginine and arginine follow analogous metabolic routes in the biosynthesis of putrescine and cadaverine respectively.     

Enzymes of N-methylputrescine biosynthesis in relation to hyoscyamine formation in transformed root cultures of Datura stramonium and Atropa belladonna

The activities of enzymes related to the biosynthesis of N-methylputrescine, a precursor of the alkaloid hyoscyamine, have been measured in root cultures of Datura stramonium L. and Atropa belladonna L. transformed with Agrobacterium rhizogenes. Ornithine -Nmethyltransferase and -N-methylornithine decafboxylase were undetectable, indicating that -N-methylornithine is an unlikely intermediate in the formation of N-methylputrescine. The activity of putrescine-N-methyltransferase (EC 2.1.1.53) was comparable to, or greater than, that of arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17). Radiolabel from dl-[5-¹⁴C]ornithine, l-[U-¹⁴C]arginine, [U-¹⁴C]agmaine and [1,4-¹⁴C]putrescine was incorporated into hyosyamine by Datura cultures. Hyoscyamine production by Datura cultures was substantially inhibited by the arginine-decarboxylase inhibitor, dl--difluoromethylarginine, but not by the corresponding ornithine-decarboxylase inhibitor, dl--difluoromethylornithine. Together with the demonstration that label was incorporated from [U-¹⁴C]agmatine, this indicates clearly that arginine is metabolised to hyoscyamine at least in part via decarboxylation to agmatine, even though a high activity of arginase (EC 3.5.3.1) was measurable under optimal conditions. The effect of unlabelled putrescine in diminishing the incorporation into hyoscyamine of label from dl-[ 5-¹⁴C] ornithine and l-[U-¹⁴C] arginine does not lend support to the theory that ornithine is metabolised via a bound, asymmetric putrescine intermediate.Abbreviations DFMA dl--difluoromethylarginine - DFMO dl--difluoromethylornithine We thank Miss E. Bent for valuable technical assistance and J. Eagles, K. Parsley and Dr. F. Mellon for mass-spectrometric analysis. We are grateful to Dr. A.J. Parr and Dr. M.J.C. Rhodes for helpful discussions. We are indebted to the Merrell Dow Research Institute, Cincinnati, Ohio, USA for supplying DFMA and DFMO.     

Biochemical strategy of sequestration of pyrrolizidine alkaloids by adults and larvae of chrysomelid leaf beetles

Tracer feeding experiments with (14)C-labeled senecionine and senecionine N-oxide were carried out to identify the biochemical mechanisms of pyrrolizidine alkaloid sequestration in the alkaloid-adapted leaf beetle Oreina cacaliae (Chrysomelidae). The taxonomically closely related mint beetle (Chrysolina coerulans) which in its life history never faces pyrrolizidine alkaloids was chosen as a 'biochemically naive' control. In C. coerulans ingestion of the two tracers resulted in a transient occurrence of low levels of radioactivity in the hemolymph (1-5% of radioactivity fed). With both tracers, up to 90% of the radioactivity recovered from the hemolymph was senecionine. This indicates reduction of the alkaloid N-oxide in the gut. Adults and larvae of O. cacaliae sequester ingested senecionine N-oxide almost unchanged in their bodies (up to 95% of sequestered total radioactivity), whereas the tertiary alkaloid is converted into a polar metabolite (up to 90% of total sequestered radioactivity). This polar metabolite, which accumulates in the hemolymph and body, was identified by LC/MS analysis as an alkaloid glycoside, most likely senecionine O-glucoside. The following mechanism of alkaloid sequestration in O. cacaliae is suggested to have developed during the evolutionary adaptation of O. cacaliae to its alkaloid containing host plant: (i) suppression of the gut specific reduction of the alkaloid N-oxides, (ii) efficient uptake of the alkaloid N-oxides, and (iii) detoxification of the tertiary alkaloids by O-glucosylation. The biochemical mechanisms of sequestration of pyrrolizidine alkaloid N-oxides in Chysomelidae leaf beetles and Lepidoptera are compared with respect to toxicity, safe storage and defensive role of the alkaloids.     

Polyamine synthesis in maize cell lines

Uptake of [¹⁴C]putrescine, [¹⁴C]arginine, and [¹⁴C]ornithine was measured in five separate callus cell lines of Zea mays. Each precursor was rapidly taken into the intracellular pool in each culture where, on the average, 25 to 50% of the total putrescine was found in a conjugated form, detected after acid hydrolysis. Half-maximal labeling of each culture was achieved in less than 1 minute. Within this time frame of precursor incorporation, only putrescine derived from arginine was conjugated, indicating that putrescine pools derived from arginine may initially be sequestered from ornithine-derived putrescine. The decarboxylase activities were measured in each culture after addition of exogenous polyamine to the growth medium to assess differential regulation of the decarboxylases. Arginine and ornithine decarboxylase activities were augmented by added polyamine, the effect on arginine decarboxylase being eightfold greater than on ornithine decarboxylase. Levels of extractable ornithine decarboxylase were consistently 15- to 100-fold higher than arginine decarboxylase, depending on the titer of extracellular polyamine. Taken as whole the results support the idea that there are distinct populations of polyamine that are initially sequestered after the decarboxylase reactions and that give rise to separate end products and possibly have separate functions.     

Evidence for arginine as the endogenous precursor of necines in heliotropium

In pyrrolizidine alkaloid-bearing Heliotropium angiospermum and H. indicum shoots exposed, in the light, to ¹⁴C-labeled CO₂ for 44 hours, the incorporation of ¹⁴C into 1,2-epoxy-1-hydroxymethylpyrrolizidine and retronecine amounted to 0.23 and 0.15%, respectively, of the total carbon assimilated. Treatment of the shoots with α-dl-difluoromethylornithine, the specific ornithine decarboxylase inhibitor, at 1 to 2 millimolar had no effect on ¹⁴C incorporation into the necines. In contrast, α-dl-difluoromethylarginine, the specific arginine decarboxylase inhibitor, prevented the incorporation of ¹⁴C into the necines of both species; the inhibitor did not affect the absolute incorporation of ¹⁴C from exogenous [1,4-¹⁴C] putrescine in either species. Thus, arginine is the only apparent endogenous precursor of the putrescine channeled into pyrrolizidines, at least in these two Heliotropium species that exhibited a relatively much higher in vitro activity of arginine decarboxylase than of ornithine decarboxylase. However, within 28 hours after administration, not only exogenous l-[5-¹⁴C]arginine, but also exogenous l-[5-¹⁴C]ornithine exhibited significant incorporation of their label into the necines, incorporation that could be partially prevented by both inhibitors. Neither inhibitor affected the rates of ¹⁴C-labeled CO₂ assimilation, transformation of labeled assimilates into ethanol-insoluble compounds, or the very high degree of conversion of the introduced amino acids into other compounds. Methodology related to alkaloid biosynthetic studies is discussed.     

Are Polyamines Transported in Etiolated Peas?

To investigate the possible transport of polyamines and their precursor amino acids, ¹⁴C-labeled putrescine, spermidine, arginine, or lysine were injected into cotyledons of 4-day etiolated pea (Pisum sativum L. cv Alaska) seedlings. After 4 hours the shoot, root, and cotyledons were homogenized and the extracted, dansylated polyamines separated by thin-layer chromatography. Little radioactivity was transported from the cotyledons when [¹⁴C]putrescine or [¹⁴C]spermidine were injected and of the radioactivity in the axis, none could be recovered as polyamines. Injection of [¹⁴C]arginine or [¹⁴C]lysine, on the other hand, led to a significant transport of radioactivity into the axis, of which a large fraction was present in the form of the diamines, putrescine or cadaverine, respectively. These results indicate that polyamines in the growing regions of etiolated pea seedlings probably arise from transport and conversion of amino acid precursors.     

Jasmonates induce over-accumulation of methylputrescine and conjugated polyamines in Hyoscyamus muticus L. root cultures

 Jasmonic acid (JA) and its methyl ester (MeJA) at concentrations ranging from 0.001 to 10 μM provoked large increases in methylputrescine levels in normal and hairy roots of Hyoscyamus muticus L.; generally, levels of free putrescine and perchloric acid-soluble conjugated putrescine, spermidine and spermine also increased dramatically. More ¹⁴C-putrescine was formed when hairy roots were incubated with labelled ornithine than with arginine; conjugated ¹⁴C-putrescine was also rapidly formed. In accord with these results, ornithine decarboxylase (EC 4.1.1.17) activity was higher than that of arginine decarboxylase (EC 4.1.1.19), and MeJA enhanced these activities about two- and fourfold, respectively. Although treatment of root cultures with jasmonates enhanced precursor (putrescine, methylputrescine) levels and accumulation of secondary metabolites such as acid-soluble conjugated di-/polyamines, it provoked only modest increases in tropane alkaloid tissue levels. Received: 24 March 1999 / Revision received: 5 October 1999 / Accepted: 26 October 1999