Unser 10. Leibniz Plant Biochemistry Symposium am 7. und 8. Mai war ein großer Erfolg. Thematisch ging es in diesem Jahr um neue Methoden und Forschungsansätze der Naturstoffchemie. Die exzellenten Vorträge über Wirkstoffe…
Omanische Heilpflanze im Fokus der Phytochemie IPB-Wissenschaftler und Partner aus Dhofar haben jüngst die omanische Heilpflanze Terminalia dhofarica unter die phytochemische Lupe genommen. Die Pflanze ist reich an…
Geschmack ist vorhersagbar: Mit FlavorMiner. FlavorMiner heißt das Tool, das IPB-Chemiker und Partner aus Kolumbien jüngst entwickelt haben. Das Programm kann, basierend auf maschinellem Lernen (KI), anhand der…
The biosynthesis of complex alkaloids in plants involves enzymes that, due to high substrate specificity, appear to have evolved solely for a role in secondary metabolism. At least one class of these enzymes, the oxidoreductases, catalyze transformations that are in some cases difficult to chemically mimick with an equivalent stereo‐ or regiospecificity and yield. Oxidoreductases are frequently catalyzing reactions that result in the formation of parent ring systems, thereby determining the class of alkaloid that a plant will produce. The oxidoreductases of alkaloid formation are a potential target for the biotechnological exploitation of medicinal plants in that they could be used for biomimetic syntheses of alkaloids. Analyzing the molecular genetics of alkaloid biosynthetic oxidations is requisite to eventual commercial application of these enzymes. To this end, a wealth of knowledge has been gained on the biochemistry of select monoterpenoid indole and isoquinoline biosynthetic pathways, and in recent years this has been complemented by molecular genetic analyses. As the nucleotide sequences of the oxidases of alkaloid synthesis become known, consensus sequences specific to select classes of enzymes can be identified. These consensus sequences will potentially facilitate the direct cloning of alkaloid biosynthetic genes without the need to purify the native enzyme for partial amino acid sequence determination or for antibody production prior to cDNA isolation. The current state of our knowledge of the biochemistry and molecular genetics of oxidases involved in alkaloid biosynthesis is reviewed herein.
Publikation
Binarová, P.; Hause, B.; Doležel, J.; Dráber, P.;Association of γ-tubulin with kinetochore/centromeric region of plant chromosomesPlant J.14751-757(1998)DOI: 10.1046/j.1365-313x.1998.00166.x
Monoclonal antibodies raised against a phylogenetically conserved peptide from the C‐terminal domain of γ‐tubulin molecule were used for immunofluorescence detection of γ‐tubulin in acentriolar mitotic spindles of plant cells. The antibodies stained kinetochore fibres along their whole length, including the close vicinity of kinetochores. After microtubule disassembly by the antimicrotubular drugs amiprophos‐methyl, oryzalin and colchicine, γ‐tubulin was found on remnants of kinetochore fibres attached to chromosomes. In cells recovering from the amiprophos‐methyl treatment, γ‐tubulin was localized with the re‐growing kinetochore microtubule fibres nucleated or captured by kinetochore/centromeric regions. On isolated chromosomes, γ‐tubulin co‐localized with α‐tubulin in the kinetochore/centromeric region. The data presented suggest that in acentriolar higher plant cells γ‐tubulin might be directly or indirectly involved in modulation and/or stabilization of kinetochore–microtubule interactions.
Publikation
Pauli, H. H.; Kutchan, T. M.;Molecular cloning and functional heterologous expression of two alleles encoding (S)‐N‐methylcoclaurine 3′‐hydroxylase (CYP80B1), a new methyl jasmonate‐inducible cytochrome P‐450‐dependent mono‐oxygenase of benzylisoquinoline alkaloid biosynthesisPlant J.13793-801(1998)DOI: 10.1046/j.1365-313X.1998.00085.x
Alkaloids derived from the tetrahydrobenzylisoquinoline alkaloid (S )‐N‐methylcoclaurine represent a vast and varied structural array of physiologically active molecules. These compounds range from the dimeric bisbenzylisoquinolines, such as the muscle relaxant (+)‐tubocurarine, to the powerful anaesthetic opiate morphine, the anti‐microbial berberine and the anti‐microbial benzo[c ]phenanthridine sanguinarine. The 3′‐hydroxylation of (S )‐N‐methylcoclaurine is a branch point that is the penultimate step in the biosynthesis of the central alkaloidal intermediate (S )‐reticuline. This study identified this enzyme as a cytochrome P‐450‐dependent mono‐oxygenase that has until now eluded attempts at identification using in vitro enzyme assays. Two alleles encoding this new enzyme (S )‐N‐methylcoclaurine 3′‐hydroxylase (CYP80B1) were isolated from a cDNA library prepared from poly(A)+ RNA isolated from methyl jasmonate‐induced cell‐suspension cultures of the California poppy Eschscholzia californica . Partial clones generated by RT–PCR with cytochrome P‐450‐specific primers were used as hybridization probes. RNA gel‐blot hybridization indicated that the transcripts for CYP80B1 accumulate in response to the addition of methyl jasmonate to the cell culture medium. Both alleles were functionally expressed in Saccharomyces cerevisiae and in Spodoptera frugiperda Sf9 cells in the presence and absence of the E. californica cytochrome P‐450 reductase. The enzyme was found to hydroxylate exclusively (S )‐N‐methylcoclaurine with a pH optimum of 7.5, temperature optimum of 35°C and a Km of 15 μm. In addition to the CYP80B1 alleles, another cytochrome P‐450 with an inducible transcript (CYP82B1) was isolated and expressed in the same manner, but was not found to be involved in alkaloid biosynthesis in this plant.
Publikation
Vignutelli, A.; Wasternack, C.; Apel, K.; Bohlmann, H.;Systemic and local induction of an Arabidopsis thionin gene by wounding and pathogensPlant J.14285-295(1998)DOI: 10.1046/j.1365-313X.1998.00117.x
The Arabidopsis Thi2.1 thionin gene was cloned and sequenced. The promoter was fused to the uidA gene and stably transformed into Arabidopsis to study its regulation. GUS expression levels correlated with the steady‐state levels of Thi2.1 mRNA, thus demonstrating that the promoter is sufficient for the regulation of the Thi2.1 gene. The sensitivity of the Thi2.1 gene to methyl jasmonate was found to be developmentally determined. Systemic and local expression could be induced by wounding and inoculation with Fusarium oxysporum f sp. matthiolae . A deletion analysis of the promoter identified a fragment of 325 bp upstream of the start codon, which appears to contain all the elements necessary for the regulation of the Thi2.1 gene. These results support the view that thionins are defence proteins, and indicate the possibility that resistance of Arabidopsis plants to necrotrophic fungal pathogens is mediated through the octadecanoid pathway.