Evidence for Arginine as the Endogenous Precursor of Necines in Heliotropium

Abstract

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.

Ornithine Decarboxylase, Polyamines, and Pyrrolizidine Alkaloids in Senecio and Crotalaria

“…This confirms the biosynthetic pathway postulated from studies with exogenous Put or its possible precursors such as Orn, Arg, Spd, or Spm introduced into Senecio plants, as discussed in Birecka et al (5). The effects of DFMO and DFMA, specific enzyme-activated irreversible inhibitors of ODC and ADC, respectively, on necine radioactivity in the treated plants showed that endogenous Arg is the only detectable precursor of Put channeled into the necines of the two borages that exhibited not only ADC but also-although low-ODC activity.…”

“…The sources of the remaining labeled compounds were the same as previously (5). Except for DFMO and DFMA, which were synthesized at Merrell Dow Research Institute, all chemicals were obtained from Sigma.…”

“…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 riddellii, S. longilobus, and C. retusa-10, 14, and 8 shoots per treatment, respectively-were exposed to H20, 2 mM DFMO, 2mM DFMA, or both inhibitors together as described previously (5). After 24 h, 2 to 4 shoots per treatment were sampled and the remaining ones, with their stems in H20, 1 mM DFMO, 1 mM DFMA, or both inhibitors together, were exposed in light to 2.5 mCi of '4C-labeled CO2 in a Plexiglas chamber as described in Birecka and Catalfamo (8).…”