ABSTRACT
Anthranilate synthase (AS, EC 4.1.3.27) catalyzes the conversion of chorismate into anthranilate, the biosynthetic precursor of both tryptophan and numerous secondary metabolites, including inducible plant defense compounds. The higher plant Ruta graveolens produces tryptophan and elicitor-inducible, anthranilate-derived alkaloids by means of two differentially expressed nuclear genes for chloroplast-localized AS alpha subunits, AS alpha 1 and AS alpha 2. Mechanisms that partition chorismate between tryptophan and inducible alkaloids thus do not entail chloroplast/cytosol separation of AS isoenzymes and yet might involve differential feedback regulation of pathway-specific AS alpha subunits. The two AS alpha isoenzymes of R. graveolens were expressed as glutathione S-transferase fusion proteins in Escherichia coli deletion mutants defective in AS activity and were purified to homogeneity. Differential sensitivity of the transformed E. coli strains toward 5-methyltryptophan, a false-feedback inhibitor of AS, was demonstrated. Characterization of affinity-purified AS alpha isoenzymes revealed that the noninducible AS alpha 2 of R. graveolens is strongly feedback inhibited by 10 microns tryptophan. In contrast, the elicitor-inducible AS alpha 1 isoenzyme is only slightly affected even by tryptophan concentrations 10-fold higher than those observed in planta. These results are consistent with the hypothesis that chorismate flux into biosynthesis of tryptophan and defense-related alkaloid biosynthesis in R. graveolens is regulated at the site of AS alpha isoenzymes at both genetic and enzymatic levels.
Subject(s)
Anthranilate Synthase/genetics , Isoenzymes/genetics , Plants/enzymology , Plants/genetics , Alkaloids/biosynthesis , Amino Acid Sequence , Amino Acids/biosynthesis , Anthranilate Synthase/metabolism , Escherichia coli/genetics , Gene Expression , Genes, Plant , Isoenzymes/metabolism , Kinetics , Molecular Sequence Data , Multigene Family , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Tryptophan/pharmacologyABSTRACT
Ruta graveolens utilizes anthranilate synthase (AS) for the synthesis both of tryptophan in primary metabolism and acridone alkaloids in secondary metabolism. AS has been purified from plants and cell cultures of R. graveolens 670- and 1700-fold, respectively. Glutamine- and ammonia-dependent AS activities were strictly co-purified in all steps. Through cDNA cloning and complementation of Escherichia coli deletion mutants defective for AS, it is shown that young Ruta plants express two genes for functional AS alpha subunits, AS alpha 1 and AS alpha 2. The data indicate that AS alpha from Ruta requires an AS beta subunit with a native molecular weight of 60-65 kDa for the glutamine-dependent reaction. Protein synthesized in vitro from cloned cDNA is processed upon import into isolated chloroplasts, indicating that mature AS alpha subunits are active in plastids in vivo. AS alpha 1 and AS alpha 2 are constitutively expressed in Ruta cell cultures, but AS alpha 1 steady-state mRNA levels are increased 100-fold 6 h subsequent to elicitation whereas AS alpha 2 expression remains constitutive. Increased AS alpha 1 transcription corresponds to elicitor-induced alkaloid accumulation. The data indicate that Ruta regulates anthranilate flux into primary and secondary metabolism through differential regulation of AS genes specific to these pathways.
Subject(s)
Anthranilate Synthase/genetics , Plants/enzymology , Plants/genetics , Amino Acid Sequence , Anthranilate Synthase/chemistry , Anthranilate Synthase/isolation & purification , Arabidopsis/genetics , Cloning, Molecular , DNA, Complementary/genetics , DNA, Plant/genetics , Enzyme Precursors/metabolism , Escherichia coli/genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Molecular Sequence Data , Molecular Weight , Plastids/enzymology , Protein Conformation , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Plant/genetics , RNA, Plant/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
(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.
ABSTRACT
Papaver somniferum L. (opium poppy) cells were elicited with a Botrytis sp. homogenate and cultured by a semi-continuous process. Elicitation induced synthesis of sanguinarine and dihydrosanguinarine. Significant release of both alkaloids into the culture medium occurred. Medium exchange at 2-day intervals enabled product recovery from spent medium and maintained culture viability. Culture growth was not inhibited by elicitor treatment necessitating sub-culture prior to re-elicitation. Re-elicited cultures displayed an increasing sensitivity (reduced growth rate, higher alkaloid yield) to the elicitor with each successive treatment and did not survive a fourth elicitation.
ABSTRACT
Mutagenicity testing of a commercial extract from Rutae Herba (Tinctura Rutae) revealed a strong effect in Salmonella typhimurium strain TA98 without S9 mix. In the presence of S9 mix only a weak response was observed. Moderate mutagenic effects were detected with and without S9 mix using strain TA100. The extract used contained the furoquinoline alkaloids dictamnine, gamma-fagarine, skimmianine, pteleine and kokusaginine, as indicated by g.c. and g.c.-m.s. analysis. The pure compounds exhibited a mutagenic activity only in the presence of S9 mix in strain TA98 as well as in strain TA100, but their specific mutagenicity differed greatly in strain TA98. We conclude that the extract studied contains different mutagenic activities and that these are only partially due to the furoquinolines present in the extract.
Subject(s)
Alkaloids/toxicity , Mutagens/isolation & purification , Plant Extracts/toxicity , Plants, Medicinal , Quinolines/toxicity , Alkaloids/isolation & purification , Mutagenicity Tests , Quinolines/isolation & purification , Salmonella typhimurium/drug effectsABSTRACT
Treatment of one cell line (No. 615) of Catharanthus roseus c.v. Little Delicata with an elicitor preparation of autoclaved and homogenized Pythium aphanidermatum culture resulted in rapid accumulation of indole alkaloids. Alkaloid formation was preceded by rapid transient increases in the extractable activities of the enzymes tryptophan decarboxylase and strictosidine synthase. The induction of these two enzyme activities occurred when cells were transferred to alkaloid production medium or treatment with fungal elicitors. Treatment of this cell line with translational or transcriptional inhibitors prevented the Pythium-induced increases of enzyme activity as well as alkaloid accumulation. When cells were transferred to alkaloid production medium the induction of strictosidine synthase activity preceded that of tryptophan decarboxylase by many hours even when cells were also treated with Pythium elicitor. Results suggested that tryptophan decarboxylase induction proceeds only when endogenous tryptamine levels were decreased by two-third. The internal cellular level of tryptamine, therefore, could regulate expression of tryptophan decarboxylase, whereas induction of strictosidine synthase or of another enzyme in the biosynthetic pathway could control channeling of tryptamine into alkaloids. The results demonstrate that fungal elicitors can be used to facilitate studies of the factors which regulate expression of indole alkaloid pathway enzymes and their ultimate pathway products.
Subject(s)
Aromatic-L-Amino-Acid Decarboxylases/biosynthesis , Carbon-Nitrogen Lyases , Plants, Medicinal/enzymology , Transferases/biosynthesis , Alkaloids/biosynthesis , Cells, Cultured , Culture Media , Cycloheximide/pharmacology , Enzyme Induction/drug effects , Indoles/biosynthesis , Pythium/physiology , Time Factors , Tryptamines/metabolismABSTRACT
Habituated and tumorous Catharanthus roseus cells grown in the absence of hormones accumulated indole alkaloids. Total alkaloids and alkaloid pattern were the same when cells were cultured in medium without hormones or in alkaloid production medium with and without indole acetic acid. Treatment of cells with Pythium homogenate as elicitor did not increase total alkaloids or change the pattern of alkaloids produced. When either habituated or tumorous cells were grown in 1B5 medium after Gamborg et al (1968) containing 2,4-dichlorophenoxyacetic acid (2,4-D), their capacity to accumulate alkaloids decreased with time. The levels of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) specific activities were constant throughout growth except when cells were exposed to 2,4-D in 1B5 medium, where enzyme activities declined in step with the decrease in alkaloid accumulation. Neither habituated nor tumorous cell suspension cultures accumulated vindoline, nor could they be induced to produce this alkaloid by any of the given treatments.
ABSTRACT
Callus and cell suspension cultures were established with shoots of the soladulcidine variety of the bittersweet Solanum dulcamara L. Plantlets were regenerated from undifferentiated callus. From mixotrophic callus as well as mixotrophic suspension cultures soladulicidine, solasodine and the corresponding neutral spirostanes tigogenin and diosgenin were isolated and identified by thin layer chromatography and mass spectrometry. Total alkaloid concentrations were about 0.2 mg/g dry weight (callus) and 0.1 mg/g dry weight (green suspension cultures). In the heterotrophic cell line only the neutral sapogenins could be detected. Alkaloid accumulation in callus of Solanum dulcamara could be enhanced by the induction of organogenesis. The shoots of the regenerated plants from the mixotrophic callus contained soladulcidine (1.6 mg/g dry weight) and tigogenin. Thus, in concentration and composition the regenerated plants equalled the source plant.
ABSTRACT
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.