ABSTRACIIn pyrrolizidine alkaloid-bearing Heliotropiwn angiospermum and H. indicum shoots exposed, in the light, to "4C-labeled CO2 for 44 hours, the incorporation of 4C 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 a-DL-difluoromethylornithine, the specific ornithine decarboxylase inhibitor, at 1 to 2 millimolar had no effect on 14C incorporation into the necines. In contrast, a-DL-difluoromethylarginine, the specific arginine decarboxylase inhibitor, prevented the incorporation of '4C into the necines of both species; the inhibitor did not affect the absolute incorporation of '4C from exogenous [1,4-'4C 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-`4Caginine, but also exogenous L-j5-'4Cjornithine 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. endogenous source(s) of Put in pyrrolizidine alkaloid-bearing Heliotropium plants, we determined in preliminary studies the in vitro activities of ODC and ADC from young leaves of four Heliotropium species (8). Although the ADC activities were lower than those found in plants not containing pyrrolizidines, the activities of ODC were much lower than those of ADC. The levels of alkaloids found in the leaves showed no significant correlation with the ADC activities.This study reports the effects of DFMO and DFMA, specific enzyme-activated irreversible inhibitors of ODC and ADC, respectively, on biosynthesis of necines from precursors formed in situ in Heliotropium angiospermum and H. indicum plants exposed to pulse labeling with '4C-C02 in light. We also used Senecio vulgaris3 obtained from California, the only population available to us at the time of the experiments.For comparison, the incorporation of labeled exogenous L-Orn and L-Arg into the necines was also investigated.In H. angiospermum, over 90% of the total alkaloid content is represented by (lp, 2,B-epoxy-la-hydroxymethyl-8a-pyrrolizidine ( Fig. 1), occurring in a nonesterified form (5); in the plants used this alkaloid represented about 95% of the total. In H. indicum, esterified retronecine amounts to about 97% of the total alkaloid (9). In the previous in vitro studies ODC and ADC extracted from both species were completely inhibited by 1 mm DFMO and DFMA, respectively.

When tested for ornithine and arginine decarboxylases, pyrrolizidine alkaloid-bearing Senecio riddellii, S. longilobus (Compositae), and Crotalaria retusa (Leguminosae) plants exhibited only ornithine decarboxylase activity. This contrasts with previous studies of four species of pyrrolizidine alkaloid-bearing Heliotropium (Boraginaceae) in which arginine decarboxylase activity was very high relative to that of ornithine decarboxylase. Unlike Heliotropium angiospermum and Heliotropium indicum, in which endogenous arginine was the only detectable precursor of putrescine channeled into pyrrolizidines, in the species studied here using difluoromethylornithine and difluoromethylarginine as the enzyme inhibitors-endogenous ornithine was the main if not the only precursor of putrescine converted into the alkaloid aminoalcohol moiety. In S.riddeliji and C. retusa at flowering, ornithine decarboxylase activity was present mainly in leaves, especially the young ones. However, other very young organs such as inflorescence and growing roots exhibited much lower or very low activities; the enzyme activity in stems was negligible. There was no correlation between the enzyme activity and polyamine or alkaloid content in either species. In both species only free polyamines were detected except for C. retusa roots and inflorescence-with relatively very high levels of these compounds-in which conjugated putrescine, spermidine, and spermine were also found; agmatine was not identified by HPLC in any plant organ except for C. retusa roots with rhizobial nodules. Organ-or age-dependent differences in the polyamine levels were small or insignificant. The highest alkaloid contents were found in young leaves and inflorescence.Our previous study on PA '- the same borages were able to decarboxylate exogenous Orn to Put that in turn was converted into necines. Thus, in vivo transformation of an exogenous putative precursor does not always indicate the compound formed in situ that normally serves as a precursor. This is especially true in the case of Put, which can derive not only from Orn but also from Arg via Agm, the product of Arg decarboxylation by ADC. Our attempt to identify the in situ precursor(s) of the Put channeled into pyrrolizidines in Senecio vulgaris were unsuccessful due to the extremely low PA content of the shoots.We report here the effects of DFMO and DFMA on necine biosynthesis in S. riddellii, S. longilobus (Compositae), and Crotalaria retusa (Leguminosae) plants. These species occasionally produce extremely high levels of PAs, up to 9 to 17% of leaf dry weight (23). In previously analyzed samples of S. riddellii and S. longilobus plants from New Mexico the PA contents were about 1.5 and 2.8%, respectively (10). Since there is little information about polyamines, including Put, ADC, and ODC activities, or the site(s) of alkaloid biosynthesis in PA-bearing plants, and since such information is available only for Heliotropium (5, 7-9), a more detailed examination of PA-bearing plants seemed important. Flowering...

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