Molecular evolution of the biosynthesis of necic acids from lycopsamine type pyrrolizidine alkaloids

DIETRICH OBER
Botanisches Institut
Abt. Biochemische Ökologie und Molekulare Evolution
Christian-Albrechts-Universität Kiel
Olshausenstraße 40
D-24098 Kiel
dober@bot.uni-kiel.de
http://www.uni-kiel.de/Botanik/

References
Phytochemistry. 2006 Jul;67(14):1493-502. Epub 2006 Jul 3. Tissue distribution and biosynthesis of 1,2-saturated pyrrolizidine alkaloids in Phalaenopsis hybrids (Orchidaceae). Frölich C, Hartmann T, Ober D.
Phalaenopsis hybrids contain two 1,2-saturated pyrrolizidine monoesters, T-phalaenopsine (necine base trachelanthamidine) and its stereoisomer Is-phalaenopsine (necine base isoretronecanol). T-Phalaenopsine is the major alkaloid accounting for more than 90% of total alkaloid. About equal amounts of alkaloid were genuinely present as free base and its N-oxide. The structures were confirmed by GC-MS. The quantitative distribution of phalaenopsine in various organs and tissues of vegetative rosette plants and flowering plants revealed alkaloid in all tissues. The highest concentrations were found in young and developing tissues (e.g., root tips and young leaves), peripheral tissues (e.g., of flower stalks) and reproductive organs (flower buds and flowers). Within flowers, parts that usually attract insect visitors (e.g., labellum with colorful crests as well as column and pollinia) show the highest alkaloid levels. Tracer feeding experiments with (14)C-labeled putrecine revealed that in rosette plants the aerial roots were the sites of phalaenopsine biosynthesis. However active biosynthesis was only observed in roots still attached to the plant but not in excised roots. There is a slow but substantial translocation of newly synthesized alkaloid from the roots to other plant organs. A long-term tracer experiment revealed that phalaenopsine shows neither turnover nor degradation. The results are discussed in the context of a polyphyletic molecular origin of the biosynthetic pathways of pyrrolizidine alkaloids in various scattered angiosperm taxa. The ecological role of the so called non-toxic 1,2-saturated pyrrolizidine alkaloids is discussed in comparison to the pro-toxic 1,2-unsaturated pyrrolizidine alkaloids. Evidence from the plant-insect interphase is presented indicating a substantial role of the 1,2-saturated alkaloids in plant and insect defense.

Ober D. Seeing double: gene duplication and diversification in plant secondary metabolism. Trends Plant Sci. 2005 Sep;10(9):444-9. Review.
Gene duplications drive the recruitment of genes for secondary metabolism. Gene copies are gradually modified to create genes with specificities and expression patterns adapted to the needs of the new pathway in which they are involved. Duplicated genes are often in tandem repeats, forming clusters within the plant genome. However, in some cases, clusters of nonhomologous genes have also been identified as forming a functional unit. The selective forces that have caused the establishment of new pathways are far from understood and might have changed repeatedly during evolution owing to the continuously changing environment. Recent data show that the way several classes of secondary compounds are scattered among species is attributable to independent recruitment and the inactivation of biosynthetic enzymes.

Anke S, Niemuller D, Moll S, Hansch R, Ober D. Polyphyletic origin of pyrrolizidine alkaloids within the Asteraceae. Evidence from differential tissue expression of homospermidine synthase. Plant Physiol. 2004 Dec;136(4):4037-47. Epub 2004 Nov 19.
The evolution of pathways within plant secondary metabolism has been studied by using the pyrrolizidine alkaloids (PAs) as a model system. PAs are constitutively produced by plants as a defense against herbivores. The occurrence of PAs is restricted to certain unrelated families within the angiosperms. Homospermidine synthase (HSS), the first specific enzyme in the biosynthesis of the necine base moiety of PAs, was originally recruited from deoxyhypusine synthase, an enzyme involved in the posttranslational activation of the eukaryotic initiation factor 5A. Recently, this gene recruitment has been shown to have occurred several times independently within the angiosperms and even twice within the Asteraceae. Here, we demonstrate that, within these two PA-producing tribes of the Asteraceae, namely Senecioneae and Eupatorieae, HSS is expressed differently despite catalyzing the same step in PA biosynthesis. Within Eupatorium cannabinum, HSS is expressed uniformly in all cells of the root cortex parenchyma, but not within the endodermis and exodermis. Within Senecio vernalis, HSS expression has been previously identified in groups of specialized cells of the endodermis and the adjacent root cortex parenchyma. This expression pattern was confirmed for Senecio jacobaea as well. Furthermore, the expression of HSS in E. cannabinum is dependent on the development of the plant, suggesting a close linkage to plant growth.

Reimann A, Nurhayati N, Backenkohler A, Ober D. Repeated evolution of the pyrrolizidine alkaloid-mediated defense system in separate angiosperm lineages.
Plant Cell. 2004 Oct;16(10):2772-84.
Species of several unrelated families within the angiosperms are able to constitutively produce pyrrolizidine alkaloids as a defense against herbivores. In pyrrolizidine alkaloid (PA) biosynthesis, homospermidine synthase (HSS) catalyzes the first specific step. HSS was recruited during angiosperm evolution from deoxyhypusine synthase (DHS), an enzyme involved in the posttranslational activation of eukaryotic initiation factor 5A. Phylogenetic analysis of 23 cDNA sequences coding for HSS and DHS of various angiosperm species revealed at least four independent recruitments of HSS from DHS: one within the Boraginaceae, one within the monocots, and two within the Asteraceae family. Furthermore, sequence analyses indicated elevated substitution rates within HSS-coding sequences after each gene duplication, with an increased level of nonsynonymous mutations. However, the contradiction between the polyphyletic origin of the first enzyme in PA biosynthesis and the structural identity of the final biosynthetic PA products needs clarification.

Nurhayati N, Ober D. Recruitment of alkaloid-specific homospermidine synthase (HSS) from ubiquitous deoxyhypusine synthase: Does Crotalaria possess a functional HSS that still has DHS activity? Phytochemistry. 2005 Jun;66(11):1346-57.
Quinolizidine alkaloids are the most prominent group of alkaloids occurring in legumes, except for many members of the tribe Crotalarieae that accumulate pyrrolizidine alkaloids (PAs). To study the evolution of PA biosynthesis as a typical pathway of plant secondary metabolism in this tribe, we have searched for a cDNA coding for homospermidine synthase (HSS), the enzyme catalyzing the first specific step in this biosynthesis. HSS was shown to have been recruited from deoxyhypusine synthase (DHS) by independent gene duplication in several different angiosperm lineages during evolution. Except for a cDNA sequence coding for the DHS of Crotalaria retusa, no data is available concerning the origin of PA biosynthesis within this tribe of the Fabaceae. In addition to several pseudogenes, we have identified one functional DHS in C. scassellatii and two in C. juncea. Despite C. juncea plants under study being devoid of PAs, we have found that the two sequences of C. juncea are different with respect to their genomic organization, their tissue-specific expression, and their biochemical activities. Supported by the branching pattern of a maximum likelihood analysis of these sequences, they have been classified as "class 1" and "class 2" DHS. It remains open whether the duplicated DHS belonging to class 2 is involved in the biosynthesis of PAs.

Ober, D. (2003) Chemical ecology of alkaloids exemplified with the pyrrolizidines.
Recent Adv. Phytochem. (J. T. Romeo et al., eds., Pergamon, Amsterdam) 37, : 203-230

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