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:
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,
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.
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
is shown on the right. The reaction pockets 1 and 2 appear to control different
steps of the reaction sequence.
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.
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.
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.
Chemotype formation in Lamiaceae. A: Assessing chemotypes in a natural
population of thyme.
Dr. Tobias Köllner
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
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.