Plant Physiol. (1987) 84, 42-46 0032-0889/87/84/0042/05/$0 1.00/0

Evidence for Arginine as the Endogenous Precursor of Necines in Heliotropium1 Received for publication August 4, 1986 and in revised form December 24, 1986

HELENA BIRECKA*, MIECZYSLAW BIRECKI, AND M. W. FROHLICH Department ofBiological Sciences, Union College, Schenectady, New York 12308

ABSTRACI endogenous source(s) of Put in pyrrolizidine alkaloid-bearing Heliotropium plants, we determined in preliminary studies the In pyrrolizidine alkaloid-bearing Heliotropiwn angiospermum and H. in vitro activities of ODC and ADC from young leaves of four indicum shoots exposed, in the light, to "4C-labeled CO2 for 44 hours, Heliotropium species (8). Although the ADC activities were lower the incorporation of 4C into 1,2-epoxy-1-hydroxymethylpyrrolizidine and than those found in plants not containing pyrrolizidines, the retronecine amounted to 0.23 and 0.15%, respectively, ofthe total carbon activities of ODC were much lower than those of ADC. The assimilated. Treatment of the shoots with a-DL-difluoromethylornithine, levels of alkaloids found in the leaves showed no significant the specific ornithine decarboxylase inhibitor, at 1 to 2 millimolar had correlation with the ADC activities. no effect on 14C incorporation into the necines. In contrast, a-DL-difluo- This study reports the effects of DFMO and DFMA, specific romethylarginine, the specific arginine decarboxylase inhibitor, prevented enzyme-activated irreversible inhibitors of ODC and ADC, re- the incorporation of '4C into the necines of both species; the inhibitor did spectively, on biosynthesis of necines from precursors formed in not affect the absolute incorporation of '4C from exogenous [1,4-'4C situ in Heliotropium angiospermum and H. indicum plants ex- putrescine in either species. Thus, arginine is the only apparent endoge- posed to pulse labeling with '4C-C02 in light. We also used nous precursor of the putrescine channeled into pyrrolizidines, at least in Senecio vulgaris3 obtained from California, the only population these two Heliotropium species that exhibited a relatively much higher available to us at the time of the experiments. in vitro activity of arginine decarboxylase than of ornithine decarboxyl- For comparison, the incorporation of labeled exogenous ase. However, within 28 hours after administration, not only exogenous L-Orn and L-Arg into the necines was also investigated. L[5-`4Caginine, but also exogenous L-j5-'4Cjornithine exhibited signif- In H. angiospermum, over 90% of the total alkaloid content icant incorporation oftheir label into the necines, incorporation that could is represented by (lp, 2,B-epoxy- la-hydroxymethyl-8a-pyrrolizi- be partially prevented by both inhibitors. Neither inhibitor affected the dine (Fig. 1), occurring in a nonesterified form (5); in the plants rates of 14C-labeled CO2 assimilation, transformation of labeled assimi- used this alkaloid represented about 95% of the total. In H. lates into ethanol-insoluble compounds, or the very high degree of con- indicum, esterified retronecine amounts to about 97% of the version ofthe introduced amino acids into other compounds. Methodology total alkaloid (9). In the previous in vitro studies ODC and ADC related to alkaloid biosynthetic studies is discussed. extracted from both species were completely inhibited by 1 mm DFMO and DFMA, respectively. MATERIALS AND METHODS Plant Material. The plants were grown in the greenhouse as previously described (6). Youngest shoots of flowering H. an- The biosynthetic pathway leading to the formation of the giospermum and H. indicum and derooted plants of S. vulgaris aminoalcohol moiety (necine) of pyrrolizidine alkaloids from at budding were used; the leaf/stem fresh weight ratio in H. two molecules of Put2 has found strong experimental support angiospermum was about 2:1, whereas in the other species it was from studies in which specifically labeled Put, Orn, Arg, sper- about 1:1. midine, or spermine were introduced into the plants and the Experiment A. The shoots were treated with H20 (control), 2 labeling pattern of the necine was analyzed after a period of 3 to mM DFMO, 2 mm DFMA, or both inhibitors together by putting 14 or more days. These studies, on Senecio magnificus (1), S. the stems in the appropriate solutions in small Erlenmeyer flasks vulgaris (1 1), and S. isatideus (12, 13, 18, 19, 21), were restricted in the light. After 24 h, 4 to 6 shoots per treatment were sampled, to retronecine (Fig. 1), the most commonly occurring necine. weighed, cut, and immediately frozen in two replicates. The Labeled exogenous Orn was also used as a precursor of retrone- remaining ones (4-6 per treatment), with their stems in H20, I cine in S. jacobaea (17) and in Crotalaria retusa (16), but the mM DFMO, 1 mM DFMA, or both inhibitors together were labeling pattern was not examined. In comparative studies, ex- exposed in light to 2.6 mCi of'4C-labeled CO2 in a plexiglass ogenous L-Orn and L-Arg exhibited rather similar absolute in- chamber as previously described in detail (4). The labeled CO2 corporations into the necine in Senecio (1, 19, 21). released together with the CO2 present in the enclosed air To assess the possible role of these two amino acids as the amounted to about 13 mg C; additional cold CO2 corresponding to 15 mg C was released 5 h after the 14C02 release. The average ' Presented at the International Union ofPure and Applied Chemistry specific radioactivity of C amounted to about 2 x 105 cpm/,ug 15th International Symposium, August 17-22, 1986, in the Hague, the C. After 44 h exposure the chamber was flushed with air and the Netherlands. plants were sampled. Only traces of radioactivity were found in 2Abbreviations: Put, putrescine; Orn, ornithine; Arg, arginine; ODC, the solutions in which the stems were immersed. ornithine decarboxylase; ADC, arginine decarboxylase; DFMO, a-DL- difluoromethylornithine; DFMA, a-DL-difluoromethylarginine; TLE, 'Voucher specimens are on deposit at the Union College Herbarium thin layer electrophoresis. (UCS). The Heliotropium collections are the same as used previously (8). 42 ARGININE: ENDOGENOUS PRECURSOR OF PYRROLIZIDINES IN HELIOTROPIUM 43 OH CH20H CH20H H [5-'4C]Orn (55 mCi/mmol); and DL-[5-'4C]Arg (12.3 mCi/

0 mmol) were obtained from Research Product International. DL- DFMO and DL-DFMA were provided by Merrell Dow Research NXt Institute; all remaining chemicals were obtained from Sigma. Retronccine 11 2 1-cpoxy- I c-hydroxyrnciylY- 8a-pyrrALizidinc RESULTS FIG. 1. Necines isolated from Heliotropium indicum (retronecine) Two exposures of plants to labeled CO2 were performed. In and H. angiospernum (1 3,2,-epoxy-la-hydroxymethyl-8a-pyrrolizi- the first, only the two species of Heliotropium were used; in the dine). second S. vulgaris plants were added, but the treatment with both inhibitors together had to be eliminated due to the size of Experiment B. Similar plants of the three species were given, the plexiglass chamber. Both experiments yielded similar results. in light, 1 ml of H20, 1 mm DFMO, or 1 mm DFMA per shoot The tables show results obtained in the second experiment. The of H. indicum and S. vulgaris and per 3 shoots of H. angiosper- highest total radioactivity was found in H. angiospermum and mum in test tubes. After the solutions were absorbed (within the lowest in H. indicum (Table I). No significant effects of 5-6 h) additional 1 ml portions of H20, 0.1 mM DFMO, or 0.1 DFMO or DFMA on the rate of CO2 assimilation or on the mM DFMA were supplied. Afterward, all shoots were given H20. transformation oflabeled assimilates into ethanol-insoluble com- Twenty-four h after the beginning of the experiments, shoots pounds were observed in any of the tested species. Both inhibi- pretreated with H20, DFMO and DFMA received 1 ml portions tors, which trailed their corresponding amino acid analogs on of 0.4 mm L-Orn or L-Arg (sulfate salts) each containing 1 ACi TLE plates, could be detected in the HC104 extracts from stems DL-t5-'4C]Orn or DL-[5-'4C]Arg (hydrochlorides), respectively. as well as from leaves of the tested plants. After the solutions of the labeled compounds were absorbed, The levels of alkaloids in the young shoots of Heliotropium H20 was added to the test tubes. In an additional experiment, (Table II) were relatively high, especially in H. angiospermum. shoots of the three species were fed H20 for 2 d or 2 mM DFMA Unfortunately, the alkaloid content of S. vulgaris was very for 24 h followed by 1 mm DFMA for the following day (as in disappointing; neither changes in the form or levels of nitrogen experiment A). Afterward, they were given 1 ml portions of 0.4 fertilizers nor increased light intensity during plant growth in- mM Put each containing 1 1sCi of 1,4-'4C]Put and tested as those creased alkaloid production in these plants. Only traces of 14C given Orn or Arg. All shoots were sampled 28 h after they were were found in the retronecine fraction of the controls shoots. supplied with the labeled compounds. In contrast, the incorporation of 14C into retronecine, and Alkaloids. Extraction, purification of alkaloids, hydrolysis of especially into epoxy-hydroxymethylpyrrolizidine in control He- retronecine esters, recovery offree retronecine, necine separation liotropium shoots was relatively high and not much lower than by TLC as well as recrystallization of the labeled necines (only the specific incorporation of exogenous Orn or Arg reported in from control and DFMO-treated Heliotropium plants) were car- the publications mentioned above. The specific radioactivities of ried out in similar ways as previously described (4). Cold retro- the necines from control and DFMO-treated plants prior to necine was obtained from monocrotaline or from alkaloids recrystallization did not differ significantly from those obtained extracted from H. indicum; cold 1,2-epoxy-hydroxymethylpyr- after two recrystallizations. DFMO did not affect the incorpora- rolizidine was extracted and purified from H. angiospermum tion of 14C into the necines in either Heliotropium species, thus plants. indicating that endogenous Orn was not a precursor, at least a ODC and ADC activities were determined in shoots sampled measurable precursor, of Put that is incorporated into pyrrolizi- immediately prior to exposure to '4CO2. Homogenization using dines. In contrast, DFMA prevented biosynthesis of the necines 200 mm phosphate buffer (pH 8.0), (NH4)2SO4 fractionation, from radioactive precursors. The slight radioactivity detected dialysis, ODC and ADC assays, and the TLE procedures to may be due to radioactive impurities and/or to the activity of quantify agmatine and/or Put formed in the reaction mixtures some ADC molecules not reached by DFMA. In the first expo- from L-[U'4C]Arg or L-[U-'4C]Orn were similar as previously sure to '4CO2, in both species, similar slight 'radioactive noise' described (7, 8); 0.1 M aminoguanidine was used to inhibit was found in DFMA treated plants as in plants treated with amine oxidases in the reaction mixtures in the assays of ODC DFMA and DFMO together. and ADC, and 20 mm Om was used to suppress arginase activity ODC and ADC activities in the control shoots of both heli- in ADC assays. otropiums (Table III) resemble those found previously in young In the case of S. vulgaris, crude extracts as well as (NH4)2SO4 leaves (8). When analyzed separately, the stems, especially in H. fractions were assayed at pH values ranging from 7.0 to 8.5 in indicum, revealed somewhat lower activities of the enzymes as the presence and absence of added aminoguanidine. compared with those found in the leaves. To examine the transformation of labeled Orn and Arg fed to In DMFA-treated shoots only traces of ADC activity were Heliotropium shoots, samples of the latter were extracted with found; ODC activity could not be detected in the DFMO-treated 6% HCI04 and the distribution ofthe radioactivity in the extracts shoots of either Heliotropium species. was determined by TLE in the same way as in the ODC and In the experiment in which [1,4-'4C]Put was introduced into ADC assays. TLE of HC104 extracts for plants exposed to '4CO2 the shoots, 28 h after its application the absolute incorporation was also performed. The RF values of Orn, Arg, and polyamines of '4C into the necines of control Heliotropium plants and those related to Put were similar to those previously reported (8). pretreated with DFMA was similar and amounted to 0.70 to TLE at pH 4.2 was performed in a cold room using a CAMAG 0.75 and 0.61 to 0.64% in H. angiospermum and H. indicum, high voltgage electrophoretic apparatus. The radioactivity was respectively. Thus, the inhibition of labeled necine biosynthesis determined using liquid scintillation system 725, Nuclear Chi- by DFMA can be clearly related to the inhibition of putrescine cago, with an efficiency of about 83 to 90% of 14C put in formation from Arg and not to an effect of DFMA on the scintillation vials in solution or in silica gel scrapped off after biosynthetic pathway leading from Put to the pyrrolizidines. TLC or TLE. At the detectability threshold of about 100 pmol/h.g fresh Chemicals. ['4C]BaCO3 (55.7 mCi/mmol); L-[U-'4C]Orn (250 weight neither ODC nor ADC activity could be detected in S. mCi/mmol); L-[U-'4C]Arg (270 mCi/mmol); DL-[1-'4C]Orn (57 vulgaris shoots regardless of the buffer pH used. mCi/mmol); and [1 ,4-'4C]Put (104.6 nCi/mmol) were purchased Since the endogenous levels of free Arg and Orn in the Heli- from New England Nuclear; DL-[I-_4C]Arg (46 mCi/mmol); DL- otropiurn leaves are low (8), relatively low amounts of the exog- 44 BIRECKA ET AL. Plant Physiol. Vol. 84, 1987 Table I. Fresh Weight and Radioactivity ofPlants Stems of the shoots were immersed in H20, 2 mM DFMO, or 2 mM DFMA starting 24 h prior to exposure to '4C-labeled CO2. During the 44 h exposure the DFMO and DFMA concentrations were decreased to I mm. Fresh Weight Radioactivity Plant Species Treatment Immediately Ethanol Ethanol before After44 h exposure soluble insoluble Total exposure fracton fraction g per shoot % dry wt x 106 cpm/gfresh wt H. angiospermum H20 0.59 0.63 20.0 136 79 215 DFMO 0.64 0.56 20.2 135 88 223 DFMA 0.58 0.63 20.2 135 79 214 H. indicum H20 1.48 1.51 17.5 46 25 71 DFMO 1.50 1.41 17.7 43 21 64 DFMA 1.71 1.52 17.8 57 33 90 S. vulgaris H20 1.23 1.45 9.1 46 35 81 DFMO 1.30 1.37 9.0 42 32 74 DFMA 1.41 1.31 9.0 42 30 72

Table II. Alkaloid Content and Radioactivity in Plants Exposed to '4C-Labeled CO2 Radioactivity of Alkaloid Content Necines Recovered Plant Species Treatment before Necine Hydrolysis after Hydrolysis fractionNeieafrTL after Ncn fe L hydrolysis ,gmol/gfresh wt % dry wt 10-3 cpm/gfresh wt % total H. angiospermum H20 24.7 1.91 438 0.203 DFMO 27.2 2.09 432 0.194 DFMA 26.7 2.04 16 0.008 H. indicum H20 12.8 12.2 1.08 288 106 0.149 DFMO 12.0 11.2 0.98 271 87 0.149 DFMA 12.2 11.4 0.99 17 4.5 0.005 S. vulgaris H20 0.50 0.42 0.07 9.1 1.1 0.0013 DFMO 0.43 0.38 0.06 10.2 1.1 0.0014 DFMA 0.45 0.39 0.07 7.0 0.7 0.0010 a 1,2-Epoxy-l-hydroxymethyl pyrrolizidine in H. angiospermum and retronecine in H. indicum and S. vulgaris; both necines with mol wt 155 contain about 62% C. Table IV. Incorporation of14C into Necines Table III. TLE-Based ODC and ADC Activities in Control Shoots of Shoots Fed Heliotropium Immediately before Exposure to "'C-labeled CO2 Speciese DL-[5-"4C]OM DL-[5-"4C]Arg In the DFMO-treated shoots no ODC activity could be detected and only traces of ADC activity were found in the DFMA-treated shoots of H20 DFMO DFMA H20 DFMO DFMA both species. % total radioactivityb ADC H. angiospermum 0.51 0.20 0.40 0.27 0.20 0.09 Plant Species' ODC H. indicum 0.42 0.21 Total Agmatine Putrescine 0.31 0.31 0.27 0.10 a In S. vulgaris the radioactivity of retronecine obtained from TLE nmol/h-gfresh wt was below 0.05% of total radioactivity. b Half of the radioactivity of H. angiospermum 0.9 7.4 6.5 0.9 DL-[5-'4C]Orn or is referred to as total radioactivity. H. indicum 0.2 12.3 8.9 3.4 DL-[5-"4C]Arg a When assayed under similar conditions using [1-'4C] or [U-'4C]Orn enous amino acids were administered. As shown in Table IV, in and Arg at 0.2 to 1 mm, extracts from S. vulgaris plants (corresponding both species, the label was incorporated into the necines not only to 50-100 mg fresh wt per reaction mixture) did not reveal any activity from the exogenous Arg, but also from the exogenous Orn. of ODC or ADC measurable either on the basis of CO2 evolution or Moreover, the absolute incorporation of 14C from Orn was even agmatine and/or putrescine formation. The lowest detectability threshold higher than that from Arg, amounting to 0.4 to 0.5% when only at 0.2 mm substrate was about 100 pmol/h.g fresh wt. the radioactivity of the L-forms of the introduced amino acids are taken into account. No incorporation ofthe label either from D[5-'4C]Om or D-[5-'4C]Arg into retronecine was found in S. isatideus (2 1). ARGININE: ENDOGENOUS PRECURSOR OF PYRROLIZIDINES IN HELIOTROPIUM 45

After TLE of the L-Orn and L-Arg solutions labeled with DL- both Heliotropium species Arg was the only measurable endog- amino acids the radioactivities detected in the Orn and Arg enous precursor of the Put incorporated into necines. No meas- bands amounted to about 95% of the total activities (Table V). urable role of endogenous Orn as a necine precursor could be Assuming that only 50% of the introduced activity labeled the detected, in spite of the fact that the plants, especially H. angio- cold L-forms ofthe amino acids and that there were no enzymic spermum, did reveal measurable, although low, in vitro activities or nonenzymic changes in the D-forms ofthe [5-'4C]amino acids, of ODC. However, it appears that exogenous Orn can yield about 90% of Orn and Arg fed to the plants had already been necines from ODC-produced Put. The observed differences be- metabolized 28 h after application. In the case ofOrn about 25% tween exogenous Orn and Arg in the incorporation oftheir label ofthis activity was found in compound(s) with RF of Arg; in the into necines may be largely due to differences in dilution factors case of Arg about 15% was detected in compound(s) with RF of during their conversion into Put, the dilution factor being much Orn. Obviously, there is no proof that any of the compounds higher for Arg due to the larger number of steps in its pathway. extracted from plants and separated by TLE are, in fact-even Obviously, with exogenous precursors, there is no control either in part-the compounds indicated for the RF in the Table. over their transport through tissues and cells or over the intra- However, the effects of DFMO and DFMA on necine radioac- cellular sites of their penetration. tivity suggest that in plants which do not use endogenous Orn as No information could be found on transformations of exoge- a measurable source of Put for pyrrolizidine biosynthesis (a) nous Orn or Arg after their introduction into a pyrrolizidine exogenous Orn may be decarboxylated in vivo by ODC to Put alkaloid-bearing plant except for the incorporation of the label which is in turn channeled into pyrrolizidines and (b) exogenous into the alkaloid. Neither could any information be found about Orn may give rise to Arg which in those plants may serve as a ODC or ADC activities in any such plant, except for the heli- precursor of pyrrolizidines. otropiums discussed here. The question of the ODC in situ involvement in metabolism DISCUSSION as well as of ADC involvement in pathways other than those In the anabolic pathway in higher plants Arg derives via leading to pyrrolizidines in the investigated borages remains open. The results obtained indicate that citrulline as the possible citrulline from Orn which in turn derives from glutamate formed in chloroplasts via N-acetylglutamate and N-acetylglutamyl-5- precursor of Put (10) channeled into pyrrolizidines, at least in the two Heliotropium may be eliminated. semialdehyde; the exact intracellular location of the enzymes species, involved is not known, beyond the fact that mitochondria play In our previous experiments with H. spathulatum (4) which contains three necines in measurable amounts, the resulting a partial role. In the catabolic pathway, membrane-separated from the anabolic pathway, Arg hydrolyzed by arginase yields changes in the total and specific radioactivities of the necines Orn which, as with exogenous Orn, may give rise to glutamyl-5- after exposure of the plants to 14C02 indicated that trachelan- semialdehyde (23). thamidine (1-hydroxymethylpyrrolizidine) is the first necine As is well known, Orn yields Put directly due to ODC, whereas formed-apparently via homospermidine (6, 13, 20)-and that Arg yields Put indirectly via agmatine, the product of ADC it may in turn be converted into supinidine (the corresponding activity, with the involvement of two additional enzymes, ag- 1,2-unsaturated aminoalcohol) and retronecine. Recently labeled matine deaminase and N-carbamyl putrescine amidohydrolase trachelanthamidine introduced into S. isatideus proved to be an (22). efficient precursor of retronecine when tested a week after its The results obtained with DFMA-treated Heliotropium plants application (14); and it yielded 15 to 20% absolute incorporation exposed to '4C02 are the first proof that endogenous Put from into retronecine in S. riddellii after 3 weeks of feeding (15). an endogenous source is the precursor of necines in a pyrrolizi- Although we could not detect trachelanthamidine either in S. dine alkaloid-bearing plant, supporting the conclusions drawn in riddellii or S. longilobus from New Mexico with alkaloid contents this respect from experiments with exogenous precursors intro- of 1.5 and 2.8% dry weight, respectively (5), we did identify duced into Senecio plants. At the same time, our experiments trachelanthamidine in H. angiospermum and H. indicum plants are a reminder that the intracellular compartmentalization of at about 1 to 4% of total alkaloid (5, 9). The formation of endogenous substrates and enzymes may determine whether a retronecine from labeled Put in H. indicum involves more steps potential indirect precursor is incorporated, especially when two than that of the 1,2-epoxy-1-hydroxymethylpyrrolizidine in H. or more possible metabolic pathways exist in a cell; such a control angiospermum. This might be one of the factors that resulted in mechanism may be ineffectual with exogenous compounds. In a lower incorporation of 14C into the necine in H. indicum with Table V. Distribution ofRadioactivity in HCl04 Extracts ofH. angiospermum Shoots Fed L-OrMl or L-Arg labeled with DL-[5-14C]Orn or DL-[5-'4C]Arg, respectively. (H. indicum shoots yielded similar results.) TLE at 500 V, 70 mamp CompouswithRF of DL-[5-'4C]OM Shoots fed DL-[5-'4C]OrM DL-[5-'4C]Arg Shoots fed DL-[5-14C]Arg Compoundssolution H20 DFMO DFMA solution H20 DFMO DFMA % total radioactivity Put 0.1 0.4 0.5 0.5 <0.1 0.9 1.1 0.9 Agmatine 0.1 0.8 0.7 0.6 Spermidine 1.0 1.5 1.3 1.5 0.4 1.7 1.8 1.9 Orn 94.9 51.4 50.4 51.8 1.0 7.5 8.5 7.8 Arg 1.4 12.8 12.7 11.5 94.8 51.9 55.1 52.2 Remaining 2.6 26.0 28.1 25.5 3.7 30.2 24.6 27.8 HC104 insoluble frac- tion 7.9 7.0 9.2 7.0 8.2 8.8 IO 6 cpm Total radioactivity 1.96 1.88 1.86 1.80 1.87 1.81 1.80 1.75 46 BIRECKA ET AL. Plant Physiol. Vol. 84, 1987 a higher in vitro ADC activity as compared to H. angiospermum chemistry 23: 991-997 a 7. BIRECKA H, AJ BITONTI, PP MCCANN 1985 Assaying ornithine and arginine exhibiting lower ADC activity. In previous studies the levels of decarboxylases in some plant species. Plant Physiol 79: 509-514 free Arg, agmatine, and Put in the young leaves ofthe two species 8. BIRECKA H, AJ BITONTI, PP MCCANN 1985 Activities ofarginine and ornithine were rather similar (8), and there was evidence that the alkaloid decarboxylases in various plant species. Plant Physiol 79: 515-519 accumulation in these leaves might be due not only to synthesis 9. CATALFAMO JL, WB MARTIN, H BIRECKA 1982 Accumulation ofalkaloids and their necines in Heliotropium curassavicum, H. spathulatum and H. indicum. in situ, but also to import from older organs (8, 9). Phytochemistry 21: 2669-2675 In relation to this investigation it is worth recalling experiments 10. CROCOMO OJ, LC BASSO, OG BRASIL 1970 Formation of N-carbamyl-putres- with lupine quinolizidine alkaloids, in which we followed the cine from citrul}ine in Sesamum. Phytochemistry 9: 1487-1489 conversion of exogenous sparteine into more oxidized quinoli- 1 1. GRUE-SORENSEN G, ID SPENSER 1982 The biosynthesis of retronecine. Can J Chem 60: 643-662 zidines in bitter white lupine plants; for comparison, we intro- 12. KHAN HA, DJ RoBINS 1981 Pyrrolizidine alkaloid biosynthesis. incorporation duced sparteine into nonalkaloidal bean plants. In both cases of '3C-labeled putrescine into retronecine. J Chem Soc Chem Commun 146- similar conversions of sparteine were found although in beans 147 they took place mostly during the few hours needed for alkaloid 13. KHAN HA, DJ ROBINS 1981 Pyrrolizidine alkaloids. Evidence for N-(4-ami- nobutyl)- 1 ,4-diaminobutane (homospermidine) as an intermediate in retro- introduction (2). Conversion of '4C-labeled lupanine into other necine biosynthesis. J Chem Soc Chem Commun 554-556 alkaloids 10 to 17 d after introduction into lupine plants was not 14. KUNEC EK, DJ ROBINS 1986 Evidence for different 1-hydroxymethylpyrroli- much different from that found after 3 d, at the first sampling zidines as intermediates in the biosynthesis ofretronecine and rosmarinecine. (3). J Chem Soc Chem Commun 251-252 15. LEETE E, J RANA 1986 Synthesis of [3,5-'4C] trachelanthamidine and {5-3H] Acknowledgments-We are grateful to Dr. Peter P. McCann for the generous isoretronecanol and their incorporation into the retronecine moiety of rid- supply of DFMO and DFMA. We also thank M. S. Arkovitz and S. Staveckis for delliine in Senecio riddellii. J Nat Prod 49: 838-84 their effective assistance during the experiments. 16. RAO PG, U ZuTsHi, A SONI, CK ATAL 1979 Studies on incorporation of 14C- labelled precursors in monocrotaline. Planta Med 35: 279-286 17. REED RL, BM REED, DR BUHLER 1985 Biosynthesis of radiolabeled pyrroli- LITERATURE CITED zidine alkaloids from Seneciojacobaea and S. vulgaris. Plant Med 5: 472 1. BALE NW, NHG CROUT 1975 Determination of the relative rates of incorpo- 18. RoBINs DJ, JR SWEENEY 1979 Pyrrolizidine alkaloids. Evidence for the in- ration of arginine and ornighine into retronecine during pyrrolizidine alka- volvement of spermidine in the biosynthesis of retronecine. J Chem Soc loid biosynthesis. Phytochemistry 14: 2617-2622. Chem Commun 120-121 2. BIRECKA H, D SZKLAREK, A MAZAN 1960 Alkaloid synthesis in derooted white 19. RoBINs DJ, JR SWEENEY 1981 Pyrrolizidine alkaloid biosynthesis. Incorpora- lupine plants. Bull Acad Pol Sci Cl. II 8: 167-173 tion of '4C-labelled precursor into retronecine. J Chem Soc Perkin I: 3083- 3. BIRECKA H, T SEBYLA 1960 Transformations of 4C-labelled lupanine in white 3086 lupine. Bull Acad Pol Sci Cl. II 8: 183-187 20. ROBINs DJ 1982 A biogenetically patterned synthesis of the pyrrolizidine 4. BIRECKA H, JL CATALFAMO 1982 Aminoalcohols of pyrrolizidine alkaloids in alkaloid trachelanthamidine. J Chem Soc Chem Commun 1289-1290 Heliotropium species. Part I. Incorporation of assimilated carbon into ami- 21. ROBINS DJ, JR SWEENEY 1983 Pyrrolizidine alkaloid biosynthesis: derivation noalcohols ofHeliotropium spathulatum. Phytochemistry 21: 2645-2651 of retronecine from L-arginine and L-ornithine. Phytochemistry 22: 457- 5. BIRECKA H, MW FROHLICH, LM GLICKMAN 1983 Free and esterified necines 459 in Heliotropium species from Mexico and Texas. Phytochemistry 22: 1167- 22. SMITH TA 1985 Polyamines. Annu Rev Plant Physiol 36: 117-143 1171 23. THOMPSON JF 1980 Arginine synthesis, proline synthesis, and related processes. 6. BIRECKA H, TE DINOLFO, WB MARTIN, MW FROHLICH 1984 Polyamines and In PK Stumpf, EE Conn, eds, The Biochemistry of Plants, Vol 5. Academic leaf senescence in pyrolizidine alkaloid-bearing Heliotropium plants. Phyto- Press, New York, pp 375-402