Dr. Jörg Degenhardt

Plants produce a huge variety of metabolites with very diverse structures and functions. Comparatively few of these compounds are required for growth and development of the plant, while others, a large group called secondary metabolites, have no direct benefit to the plant but play a role in the interaction of the plant with its environment. The secondary metabolites vary strongly among plant taxa, providing each plant species with a diverse and probably unique blend of compounds.  It is the goal of my research to understand how the diversity of natural metabolites has evolved and how these compounds have been recruited for their ecological functions. In particular, I would like to answer the following questions:

 ·    How has the high diversity of secondary products arisen in the course of evolution and how were these compounds recruited for plant defense or plant-insect signaling?

 ·    How do natural plant populations acquire and sustain several stable chemotypes that differ in their terpene compositions and defense mechanisms?

 ·    How do plants detect biotic and abiotic stress and respond with the biosynthesis of specific compounds?

The studies in my group are focused on terpenes which form the largest group of plant products with 30,000 different structures.  Many of these terpenes have an essential role in plants, including hormones (gibberellins and abscisic acid), membrane components (sterols), participating in electron transfer (ubiquinone and plastoquinone) and pigments (carotenoids). Other terpenes are apparently not vital for plant growth or development but form oils and resins that are part of the defense against other organisms. More recent is the discovery of volatile terpenes that act as signals influencing the behaviour of insects and other organisms. However, the functional role of most terpenes still needs to be determined.

a) The evolution of terpene diversity in maize and related grasses

Terpenes are an ideal system to study structural diversity because of their straightforward biosynthesis from activated five-carbon units. Of the >100 structurally diverse terpenes produced in maize, most are sesquiterpene hydrocarbons which are formed from three five-carbon units. In  detailed analysis of the maize sesquiterpene hydrocarbons, we showed that the terpenes occur in groups which each have a particular distribution within the plant and a distinct composition of terpenes (Figure 1).

Figure 1: The sesquiterpene hydrocarbons of a maize seedling can be divided in three differentially regulated groups of which group A is induced by herbivory, B is ubiquitous and C is only found in the roots. The compounds are: 1: unknown, 2: α-copaene, 3: (E)-β-caryophyllene, 4: (E)-α-bergamotene, 5: sesquisabinene A, 6: (E)-β-farnesene, 7: unknown, 8: germacrene D, 9: zingiberene, 10: α-muurolene, 11: unknown, 12: β-bisabolene, 13: δ-cadinene, 14: β-sesquiphellandrene.

 

The enzyme class responsible for most of the diversification are the terpene synthases which are encoded in large gene families. The comparison of terpene synthase structure-function relationships is therefore an excellent tool to study evolutionary processes. We identified the terpene synthase gene family from maize and characterized their activity by heterologous expression, gas chromatography with isotopic tracers, mass spectrometry and nuclear magnetic resonance (Figure 2 A). Most terpene synthases are multiproduct enzymes producing mixtures of up to forty different terpene compounds. Mapping and phylogenetic analysis of the maize terpene synthase family indicates a common origin and several sub-families. We could demonstrate that two of the terpene synthase genes are the result of a tandem duplication event about three million years ago. Subsequently, four nucleotides mutated between the two genes and thereby the enzymatic activity was altered (Figure 2 B, C).

Figure 2: Within the terpene synthase gene family, genes have duplicated and diverged to encode enzymes with different activity. A: Dendrogram analysis of maize terpene synthase gene family. B: Proposed model of the TPS4 active site cavity. The four amino acids in the bottom of the active site that differ between TPS4 and TPS5 are shown. C: Amino acid changes were introduced sequentially into the active site of TPS5 to alter the active site sequence motif ‘SIGAN’ into ‘TIATI’ as found in TPS4. The activity of the mutated enzymes was determined after heterologous expression in E. coli. 1: 7-epi-sesquithujene, 2: sesquithujene, 3: (Z)-a-bergamotene, 4: (E)-α-bergamotene, 5: sesquisabinene B, 6: sesquisabinene A, 7: (E)-β-farnesene, 8: (S)-β-bisabolene. From: Köllner et al. (2004) Plant Cell 16, 1115-1131
 

        The unique catalytic mechanism of terpene synthases enables these enzymes to form multiple products. To elucidate the reaction mechanism, we study structure-function relationships within the active site of the enzyme. Comparisons of closely related enzymes and directed alterations of single amino acids by in vitro mutagenesis helped us to identify structures responsible for the formation of multiple products (Figure 3). We identified several amino acids in the active center of the enzyme that determine product specificity and identified two catalytic pockets that appear to catalyze partial steps of the reaction. The goal of this study is to understand these fascinating reaction mechanisms and to engineer terpene synthases with a specific terpene spectrum. Furthermore, the phylogenetic analysis of structure function-relationships within maize and closely related grasses will indicate which evolutionary processes have shaped this gene family and yield to an understanding of its function.

 

Figure 3: Model of the maize terpene synthase TPS4 active site with the proposed positions of the (E,E)-farnesyl diphosphate substrate. The proposed reaction pathway of the two main products of TPS4, 7-epi-sesquithujene and (S)-β-bisabolene is shown on the right. The reaction pockets 1 and 2 appear to control different steps of the reaction sequence.
From: Köllner et al., Archives Biochemistry and Biophysics, in press.

 

b) Discovering the roles of terpenes in plant defense

Plants do not only accumulate terpenes for herbivore defense, but also emit complex volatile blends in response to herbivory, fungal attack and many other biotic and abiotic stresses. These terpene-containing volatiles attract natural enemies of the attacking herbivores but due to the complexity of these volatile blends, it is difficult to attribute a specific function to a specific terpene.

      We were able to show that maize roots damaged by the maize pest Diabrotica virgifera emit the sesquiterpene (E)-β-caryophyllene which attracts entomopathogenic nematodes. These nematodes attack the larvae of D. virgifera and thereby benefit the maize plant (Figure 4A). Above ground, the herbivory by lepidopteran larvae induces a mixture of volatiles that is highly attractive to females of various parasitic wasps (Figure 4B). We identify the terpene synthases that produce the herbivore-induced terpenes (Figure 1) and utilize mutants, transgenic plants and transposon insertion lines to study the importance of these terpene signals for plant defense. The impact of these defenses on plant fitness and its natural environment can be tested in field experiments. Plants that have an increased defense capability may have promise for agricultural use.

 Figure 4: The role of terpenes as signals in plant defense. A: Volatile terpenes released form the leaves of a maize plant after attack of a lepidopteran larvae attract parasitic wasps that are natural enemies of the lepidopteran larvae. B: Feeding of Diabrotica virgifera on maize roots (not shown) attracts entomopathogenic nematodes that attack the larvae of D. virgifera. Photos by Ted Turlings. From: Rasman et al., (2005) Nature 434: 732-737).

c) Regulation of plant natural product biosynthesis

Detailed studies have demonstrated how fungal elicitors trigger specific plant defense responses, but little is known how herbivores can trigger the direct and indirect defenses of the plant. The family of terpene synthases is well suited to study the last steps of the complex herbivore-induced signal transduction cascades (Figure 5). The comparison of promoter sequences will allow the identification of elements involved in herbivory-mediated gene expression and the characterization of trans-binding factors that interact with these elements. Our feeding experiments with potential intermediates of the signaling cascade have already demonstrated the existence of several parallel signaling pathways with different kinetics. Microarray data, differential displays of gene activity and proteomic analysis of herbivore-induced maize plants will represent the alterations in gene expression that are part of plant signaling.
 

Figure 5: Terpene synthase expression is regulated by herbivory. RNA hybridization assays show that the transcript levels of the terpene synthases tps1 and tps2 are elevated after herbivory. However, the induction of the two genes follows different kinetics. The lowest panel shows the 18S rRNA as loading control.
From: Schnee et al., (2002) Plant Physiology 130, 2049-2060.
 

d) The formation of Chemotypes in Thymus and Origanum

Terpene composition often shows substantial qualitative and quantitative differences within a single species. Natural populations of the Lamiaceae thyme and oregano consist of several chemotypes that are defined by their terpene content. The chemotypes differ in their resistance to particular herbivores and appear to be localized in the environments with the herbivore communities that they are best defended against. The goal of this study is to understand the evolution of chemotypes and the genetic mechanisms that maintain the distinct chemotypes in mixed populations Classical genetic studies on thymus demonstrated an epistatic series of six loci that define each of the chemotypes. We extend these studies towards the molecular genetics and biochemistry of chemotype formation with the aim to characterize terpene biosynthesis and the mechanisms that regulate chemotype formation.

Figure 6: Chemotype formation in Lamiaceae. A: Assessing chemotypes in a natural population of thyme.
B: Lamiaceae terpene oils are formed in glandular trichomes on the leaf surface. Clockwise: Origanum leaf, leaf surface with glandular trichomes, close up of a glandular trichome, an isolated glandular trichome with eight gland cells (Photos Christoph Crocoll).

Group members:

Dr. Tobias Köllner
Julia Asbach (supported by EU 5^th framework programme INTESY)
Christoph Crocoll (supported by EU 5^th framework programme INTESY)
Anna Fontana (International Max Planck Research School)
Claudia Lenk
Natalia Rauch (technical assistance)

Curriculum vitae

1984 - 1990         Studies of Biology at Ruhr-Universität Bochum, Germany. Masters thesis on 'Developmental expression pattern of chloroplast and nuclear genes in Arabidopsis thaliana‘

1991 - 1995         PhD thesis at University of California at Los Angeles ‘Regulation of nuclear genes by the phytochrome system: Studies on the promoters of the rbcS and Lhcb genes in Lemna gibba L.‘

1993 - 1994         Fellowship by German Academic Exchange Service (DAAD)

1995                   Doctoral defense to obtain Ph.D. degree in Biology at University of Bochum

1995 - 1999         Postdoctoral Fellow at Cornell University und  Boyce Thompson Institute

1999 - 2008         Research group leader at Max Planck Institute for Chemical Ecology

2001 - 2003         Habilitationsstipendium Claussen-Simon Stiftung

2007                   Habilitation for the field Botany at Friedrich-Schiller-Universität Jena

April 2008            Professor for Pharmaceutical Biotechnology (W3) at Martin-Luther-Universität Halle-Wittenberg

Scientific Grants

2004-2008  Priority Programme SPP 1152 “Evolution of Metabolic Diversity“ of the German Science Foundation (DFG).

2002-2006  Grant by European Union within Framework 5 "Quality of Life and Management of Living Resources": I

2001-2004  Grant of the German Department of Education and Research (BMBF) on Safety and Monitoring of Genetically Modified Plants.

Recent Publications