Heliotropium1 Received for Publication August 4, 1986 and in Revised Form December 24, 1986

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.

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