Distribution of autogenous and host-derived chemical defenses inOreina leaf beetles (Coleoptera: Chrysomelidae).

The pronotal and elytral defensive secretions of 10Oreina species were analyzed. Species feeding on Apiaceae, i.e.,O. frigida andO. viridis, or on Cardueae (Asteraceae), i.e.,O. bidentata, O. coerulea, andO. virgulata, produce species-specific complex mixtures of autogenous cardenolides.O. melanocephala, which feeds onDoronicum clusii (Senecioneae, Asteraceae), devoid of pyrrolizidine alkaloids (PAs) in its leaves, secretes, at best, traces of cardenolides. Sequestration of host-plant PAs was observed in all the other species when feeding on Senecioneae containing these alkaloids in their leaves.O. cacaliae is the only species that secretes host-derived PA N-oxides and no autogenous cardenolides. Differences were observed in the secretions of specimens collected in various localities, because of local differences in the vegetation. The other species, such asO. elongata, O. intricata, andO. speciosissima, have a mixed defensive strategy and are able both to synthesize de novo cardenolides and to sequester plant PA N-oxides. This allows a great flexibility in defense, especially inO. elongata andO. speciosissima, which feed on both PA and non-PA plants. Populations of these species were found exclusively producing cardenolides, or exclusively sequestering PA N-oxides, or still doing both, depending on the local availability of food-plants. Differences were observed between species in their ability to sequester different plant PA N-oxides and to transform them. Therefore sympatric species demonstrate differences in the composition of their host-derived secretions, also resulting from differences in host-plant preference. Finally, within-population individual differences were observed because of local plant heterogeneity in PAs. To some extent these intrapopulation variations in chemical defense are tempered by mixing diet and by the long-term storage of PA N-oxides in the insect body that are used to refill the defensive glands.

Sequestration of ingested [(14)C]senecionineN-oxide in the exocrine defensive secretions of chrysomelid beetles.

Oreina cacaliae (Chrysomelidae) sequesters in its elytral and pronotal defensive secretion theN-oxides of pyrrolizidine alkaloids (PAN-oxides) from its food plantAdenostyles alliariae (Asteraceae). [(14)C]SenecionineN-oxide was applied for detailed studies of PAN-oxide sequestration. An average of 11.4% of total radioactivity is taken up by individual beetles which had received [(14)C]senecionineN-oxide with their food leaves 8 days before. An average of 28.9% of the ingested radioactivity could be recovered from the defensive secretions collected twice, i.e., 5 and 8 days after tracer feeding. The tracer transfer into the secretion seems to be a slow but progressive process as indicated by the high percentage of tracer still recovered from the secretion sampled after 8 days. Chromatographic analysis revealed that [(14)C]senecionineN-oxide is the only labeled compound in the defensive secretion. Beetles that fed on tertiary [(14)C]senecionine sequestered only trace amounts of radioactivity (exclusively present as labeled IV-oxide) in their secretions.O. speciosissima, a species also adapted to PA containing food plants, was shown to sequester [(14)C]senecionineN-oxide with the same efficiency asO. cacaliae. O. bifrons, a specialist feeding onChaerophyllum hirsutum (Apiaceae), rejected PA treated leaf samples already at very low PA concentrations (10 nmol/leaf piece). In bothO. cacaliae andO. speciosissima, [(14)C]senecionineN-oxide applied by injection into the hemolymph is rapidly transferred into the glands.O. bifrons, not adapted to pyrrolizidine alkaloid containing plants was unable to sequester [(14)C]-senecionineN- oxide in the secretion but rapidly eliminated the tracer with the frass. Again, only traces of labeled [(14)C]senecionineN-oxide were found in the defensive secretions of the two PA adapted species if labeled senecionine was injected. It is suggested that the beetles are adapted to theN-oxide form of PAs, similarly as their food plants, and that they lack the ability to efficientlyN-oxidize tertiary PAs. No indication forde novo PA synthesis by the beetles was found in tracer feeding experiments with the biogenetic PA precursor putrescine.

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

(14)C-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 [(14)C]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 [(14)C]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.

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 (14)C-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 Km-values for sen Nox of 53 µM and 310 µM 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 µM, 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.

Elicitor-Induced Accumulation of Acridone Alkaloid Epoxides in Ruta graveolens Suspension Cultures.

Light and dark grown suspension cultures of RUTA GRAVEOLENS produced small amounts (1-50 microg/g dry weight) of the antimicrobial alkaloids rutacridone epoxide and hydroxyrutacridone epoxide. Acrid-one epoxide accumulation could be increased up to a 100 fold within 72 h by elicitation through addition of a suspension of either living free or immobilized yeasts, of dead RHODOTORULA RUBRA cells or crude cell wall fraction. Chitosan and alginate, exhibiting elicitor properties in other systems, scarcely had an effect, arachidonic acid and elaidic acid were ineffective. The elicitors of acridone epoxide accumulation did not effect an increase of rutacridone in RUTA cultures.

Plant NAD-dependent glutamate dehydrogenase. Purification, molecular properties and metal ion activation of the enzymes from Lemna minor and Pisum sativum.

Glutamate dehydrogenase (L-glutamate: NAD+ oxidoreductase (deaminating) EC 1.4.1.2) has been purified to homogeneity from Lemna minor and seeds of Pisum sativum. As established by polyacrylamide gel electrophoresis the Pisum-enzyme constitutes a multiple pattern of seven charge isoenzymes whereas the Lemna enzyme shows one single protein band. Molecular weights of 230 000 were calculated for both enzymes by sedimentation equilibrium measurements. (Pisum-enzyme) and comparative gel filtration (Lemna-enzyme). Sodium dodecyl sulfate gel electrophoresis and electron microscopic observations revealed that both enzymes are composed of four identical subunits (molecular weight 58 500) arranged in a tetraedric structure. The amino acid compositions of both enzymes are similar to those of various hexameric glutamate dehydrogenases. The N-terminal amino acid of the Pisum-enzyme is alanine. Both enzymes require Ca2+ for maximal catalytic activity. For the Lemna-enzyme the K0.5 values for Ca2+ are 22 microM (NAD+-dependent reaction), respectively. Ca2+ which to some extent can be replaced by Zn2+ does not affect the enzyme aggregation but seems to govern a reversible equilibrium between catalytically active and inactive enzyme forms.

Role of mitochondrial glutamate dehydrogenase in the reassimilation of ammonia produced by glycine serine transformation.

The ability of isolated pea-shoot mitochondria conditioned to incorporate ammonia into glutamate to reassimilate endogenously produced ammonia from glycine transformation was investigated. In the presence of 1 mM to 20 mM glycine less than 15% of the ammonia liberated was found to be incorporated into glutamate. Thus, a prominent role of mitochondrial glutamate dehydrogenase in the reassimilation of intramitochondrially produced ammonia can be excluded.