IFIB – Interfakultäres Institut für Biochemie


Diversity and Evolution of of Plant Secondary Metabolism


Since thousands of years plants are a source of nutrients, energy supply or material for construction of houses and furniture for humans. They provide also colors, flavors, fibers and pharmaceuticals, which is due to the presence of so-called “secondary metabolites” (=natural products) in plants. 50 % of all pharmaceuticals newly introduced in therapy are extracted from plants, are derived from the natural compound or the compounds served as leader for the chemists to design new more efficient drugs (Kubinyi, 2004, ÖAZ 23, 1112 ff.). In former times natural products were regarded as waste of plant metabolism. Nowadays, it is well accepted that secondary metabolites are very important for the survival of a plant in its environment. They are only “secondary” with respect to be produced from precursors as well as by enzymes developed from those of the primary metabolism. Secondary metabolites are not always evenly distributed in the plant kingdom. Therefore, they can be used as chemosystematic markers to establish plant phylogeny and to study the evolution of the biosynthetic pathways in the appropriate plant families.

Many secondary metabolites contain chiral C-atoms. In contrast to normal synthesis by the chemist, in nature, however, only one single enantiomer and not a racemic mixture accumulates. The “Contergan Desaster” opened our eyes for the importance of chiral pure pharmaceuticals. Only a few plant species are known to accumulate different enantiomers of the same compound in different species or even different organs of the same plant. The ecological importance and the factors which led to the selection of a special enantiomer in evolution are mostly unknown.

An important part of my research focuses on understanding the enantiospecific biosynthesis of natural compounds. The lignan biosynthesis serves as a model. Lignans are phenolic compounds which can be found in mosses and ferns but especially in higher plants. Most of the lignans are optically active. They are probably involved in plant defense against pathogens and herbivores. Podophyllotoxin (R,R-configuration at C-atom 8 and 8’) is the most important lignan used in medicine because its semisynthetic derivatives (e.g. etopophos®) are used in cancer therapy. Secoisolariciresinol diglucoside (possessing S,S-configuration) which accumulates in seeds of flax (Linum usitatissimum) is converted to enterodiol and enterolactone in the human intestinal tract. These compounds show high structural similarity to estrogen. They are encountered as phytoestrogens and are claimed to protect against cancer types influenced by estrogens like prostate and breast cancer. After being used as a source for oil and fibers for more than ten thousand years the high concentration of secoisolariciresinol diglucoside has brought Linum usitatissimum again in the focus of research.

Research Topics

I. Plant cell and organ cultures

Many natural compounds are isolated from wild grown plants, which often carries the danger of extinction of the plant species. Plant cell and organ cultures can be used as an alternative. The natural source for podophyllotoxin, Podophyllum hexandrum, is an endangered species. Therefore, one focus of my research is to establish in vitro systems to produce podophyllotoxin and other useful lignans. In addition, the tissue and organ cultures of lignan producing plants are useful and always available sources for our investigations of the lignan biosynthesis.

II. Cloning of genes and biochemical characterization of the enzymes involved in the biosynthesis of lignans to understand the evolutionary mechanisms underlying the chemical variety of lignan structures

Up to now most steps in lignan biosynthesis are either completely unknown or at least the responsible genes are not cloned yet. The phytochemical characterization of the lignin content in different Linum species under different growth conditions and stages together with the technical progress in genome sequencing can help us to identify new candidate genes for the biosynthesis of lignans.

At the moment we are investigating the molecular basis of the structural diversity in lignan biosynthesis by comparison of key biosynthetic steps so far they are known. The pinoresinol lariciresinol reductase (PLR) is one of the best characterized enzymes involved in the early steps of lignan biosynthetic pathways up to now. We cloned a variety of genes encoding PLRs and enzymes with similar functions forming a protein family named PIP-reductases. We investigate how in evolution gene duplication and mutagenesis could lead to the high variability of functions within this reductase family by biochemical characterization of recombinant enzymes, protein structure elucidation and mutation analysis.

III. Stereochemistry in lignan biosynthesis: molecular basis and evolution

Enantiospecificity has an important meaning in the biosynthesis of natural products since almost all natural products can only be found with one stereochemistry. Lignans are an exception because the different stereoisomers of at least simple lignans occur in parallel in the plant kingdom, even in the same plant species.

The enantiomeric composition of the lignans in Linum species is determined within the first steps shared within their biosynthetic pathways. Therefore my research focused first on the evolution of this enzyme and its enantiospecificity. The biochemical characterization of the heterologously expressed enzymes, structural biology and a mutagenesis approach let us understand what in the enzyme is responsible for the differences in enantiospecificity.

IV. Genetic manipulation of the lignan biosynthesis

We have established a transformation protocol for shoot cultures of different Linum species with Agrobacterium rhizogenes. The resulting „hairy roots“ accumulated in most cases the same lignans as the plant, its cell cultures or its roots, but often in higher amounts. Therefore, hairy roots can serve as a production system for lignans of different structural types.

We used the same system to prove the involvement of candidate genes in the biosynthesis of a certain lignan by introduction of the appropriate RNAi constructs. Our RNAi-lines with interrupted lignan biosynthesis in the early steps can be used to feed possible intermediates to show their involvement in lignan biosynthesis, too.

The system is also useful to investigate the role of genes involved in the regulation of lignan accumualtion like transcription factors. Such regulatory genes could be cloned by integrated molecular biological methods (see paragraph II).

Finally, the system is useful to manipulate the lignan biosynthesis by the heterologous expression of genes yielding higher amounts of a desired lignan and even new lignan structures.