Research Group Biochemical Ecology and Molecular Evolution

Evolution of enzymes involved in secondary metabolism of plants (Group E. Kaltenegger)

Evolution and biodiversity are two closely entangled fields of biology. Simply spoken - understanding evolutionary processes that led to today’s biodiversity can guide future perspectives to maintain the diversity in the ecosystems surrounding our daily life. Adaptation - like the development of resistance of pathogens (plant or human) – as one central driver of biodiversity, can only be properly understood in the light of evolution. And given the cellular basis of life, studying evolution should ultimately begin with the evolutionary dynamics of proteins. And this is exactly what this group is aiming for.

We use the tropic Morning Glories (Convolvulaceae), which are known for their beautiful flowers, as model organisms. Various species of this plant family produce specific plant secondary metabolites, namely the pyrrolizidine and tropane alkaloids (PAs and TAs). Both classes of alkaloids function in the plants defence against herbivores. While TAs are found in a broad range of species, PAs only occur in individual species. Thus, we postulate that TA biosynthesis is older compared to PA biosynthesis. Of note, both biosynthetic pathway show some striking similarities.

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The metabolic enzymes, which are involved in alkaloid biosynthesis, are excellent models to study evolutionary dynamics of proteins. As part of secondary metabolism, they are subject to rapid changes, because plants should deal with an army of herbivores, which themselves adapt to the plants defense – a classic ecologic phenomenon described as the “plant-herbivore arms race”. Based on this phenomenon, only plants that evolve through mutations new traits to warn of herbivores will survive.

One special type of mutations is central to our research: gene duplication. Many of the above-mentioned enzymes, that are involved in alkaloid biosynthesis, originated from a duplication. We are particularly interested in the following questions: how can duplicated genes gain new functions? Which molecular mechanisms are shaping a duplicated gene? Presently, by studying the evolution of the PA-biosynthesis specific homospermidine synthase (HSS) and the TA-biosynthesis specific putrescin-N-methyltransferase (PMT) within the Morning glories, we are trying to answer this question. Of note, both genes originated by duplication and gained a new function. Step by step we are now reconstructing HSS and PMT evolution in the ancestors of the today living Morning Glory species. With this approach, we aim to identify the mutations, which were involved in the change of function.

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Scientific background

Gene duplication

Gene duplication is a very central mechanism of evolution. Among “de novo” gene birth, gene duplication provides the genetic raw material for the evolutionary playground. Duplication can affect single genes (small scale duplications) or large fragments up to whole genomes. Generalized, a duplicated gene can experience three different fates: a) it accumulates deleterious mutations, becomes a pseudogene and finally gets purged from the genome (nonfunctionalization), b) the functions of the ancestor are divided so that both copies remain functional, but both copies together are necessary to maintain the ancestral function (subfunctionalization), and c) it can gain a new function (neofunctionalization) via beneficial mutations.

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Which of these three different outcomes eventually awaits a duplicated gene is studied in detail to finally understand which molecular factors shape the fate of a duplicated gene. Such factors can be: expression level or the number of interaction partners of the gene product. For example, one hypothesis is, that genes encoding proteins that interact tightly with many other proteins (e.g. proteins that form a complex) are less often duplicated. Furthermore, the quaternary protein structure may also affect to the duplicability of a gene. But apart from the general duplicability of a gene, the evolutionary trajectory of a how a gene adopted a new function and which mutational steps were involved are most interesting.

Up to date we could show that within the Morning Glories a single gene duplication in the early evolution of this plant family provided the gene material for HSS evolution. Its new function in PA biosynthesis, which involves basically a change in substrate specificity, however evolved possibly in parallel in the two PA-producing genera of this family, namely Merremia und Ipomoea. We will combine bioinformatic tools and biochemical characterization of promising candidate proteins to test this scenario.

Project-, Bachelor-, and Master theses

We are looking for highly motivated students who wants to study protein evolution in our group. We are offering a range of projects, starting from projects for a thesis as B.A. and M.A. student as well as internships for B.A. and M.A. students. Previous knowledge about “secondary metabolism of pants” and experience in molecular biology are helpful. As successful candidate, you will apply standard techniques of molecular biology as mentioned below. Furthermore, you will get an insight into evolutionary aspects of biology.

Please contact me, if you are interested! (ekaltenegger@bot.uni-kiel.de)

Research methods

We are using:

  • Molecular methods
    • DNA- and RNA-isolation
    • Diverse PCR-based techniques
    • Cloning
    • Heterologous expression and protein purification
    • Activity assays of proteins
  • Sequence analyses including phylogenetic analyses

Publications Dr. Kaltenegger

  • Kaltenegger, E., Ober, D. (2015). Paralogue interference affects the dynamics after gene duplication. Trends in Plant Science, 20: 814-821. PMID: 26638775, DOI: 10.1016/j.tplants.2015.10.003
  • Irmer, S., Podzun, N., Langel, D., Heidemann, F., Kaltenegger, E., Schemmerling, B., Geilfus, C.-M., Zörb, C., Ober, D. (2015). A new aspect of plant-rhizobia interaction - alkaloid biosynthesis in Crotalaria depends on nodulation. Proc. Natl. Acad. Sci. U.S.A., 112: 4164-4169. Pubmed, DOI: 10.1073/pnas.1423457112
  • Kaltenegger, E., Eich, E., Ober, D. (2013). Evolution of homospermidine synthase in the Convolvulaceae – a story of gene duplication, gene loss, and periods of various selection pressures. Plant Cell, 25: 1213-1227 PubMed, , OpenAccess
  • Ober, D., Kaltenegger, E. (2009). Pyrrolizidine alkaloid biosynthesis, evolution of a pathway in plant secondary metabolism. Phytochemistry, 70: 1687-1695 PubMed
  • Anke, S., Gondé, D., Kaltenegger, E., Hänsch, R., Theuring, C., Ober, D. (2008). Pyrrolizidine alkaloid biosynthesis in Phalaenopsis orchids: Developmental expression of alkaloid-specific homospermidine synthase in root tips and young flower buds. Plant Physiology 148 : 751-760. PubMed
  • Schinnerl, J., Kaltenegger, E., Pacher, T., Vajrodaya, S., Hofer, O., Greger, H. (2005). New Pyrrolo[1,2-a]azepine Type Alkaloids from Stemona and Stichoneuron (Stemonaceae). Chemical Monthly. 9, 1671-1680. Springer
  • Seger, C.; Mereiter, K.; Kaltenegger, E.; Pacher, T.; Greger H.; Hofer, O. (2004). Two Pyrrolo[1,2-a]azepine Type Alkaloids from Stemona collinsae Craib: Structure Elucidations, Relationship to Asparagamine A, and a new Biogenetic Concept of their Formation. Chemistry & Biodiversity (Helvetica Chimica Acta). 1, 265-279. PubMed
  • Kaltenegger, E.; Brem, B.; Mereiter, K.; Kalchhauser, H.; Kahlig, H.; Hofer, O.; Vajrodaya, S.; Greger, H. (2003). Insecticidal pyrido[1,2-a]azepine alkaloids and related derivatives from Stemona species. Phytochemistry (Elsevier), 63, 803-816. PubMed