The evolution of mono- and sesquiterpene diversity in maize
JÖRG DEGENHARDT
JONATHAN GERSHENZON
Max-Planck-Institut
für Chemische Ökologie
Beutenberg-Campus
Winzerlaer Strasse 10
D-07745 Jena
degenhardt@ice.mpg.de
http://www.ice.mpg.de/
Terpenes are the largest group of plant secondary compounds, comprising about 30,000 structures that play numerous roles in primary metabolism and ecological interactions. Their large structural and functional diversity make them an excellent model to study chemical diversity in plants. The key step of terpene biosynthesis is catalyzed by the enzyme class of terpene synthases. Terpene synthases employ an electrophilic reaction mechanism that is able to form the large diversity of terpene products. In all plant species studied so far, terpene synthases are encoded by a large family of genes that share a common evolutionary origin and evolved to form complex terpene blends. Thus, this gene family is valuable tool to study the structural and functional diversification of these genes that results in the formation of a large variety of products.
The project started with the characterization of the terpene synthases gene family in maize. After biochemical characterization of the encoded enzymes and the analysis of their terpene products, we wanted to learn about possible correlations between the catalytic activity and the structure of the active center of the enzyme. We focused our studies on the maize terpene synthase TPS4 which produces a complex mixture of 14 different olefinic sesquiterpenes. To understand the complex TPS4 reaction mechanism, we modeled the active site cavity and conducted docking simulations with the substrate farnesyl diphosphate (FPP), several predicted carbocation intermediates, and the final reaction products. The model suggests that discrete steps of the reaction sequence are controlled by two different active site pockets, with the conformational change of the bisabolyl cation intermediate causing a shift from one pocket to the other. Site-directed mutagenesis and measurements of mutant activity in the presence of (E,E)- and (Z,E)-farnesyl diphosphate as substrates were employed to test this model. Amino acid alterations in pocket I indicated that early steps of the catalytic process up to the formation of the monocyclic bisabolyl cation are probably localized in this compartment. Mutations in pocket II primarily inhibited the formation of bicylic compounds, suggesting that secondary cyclizations of the bisabolyl cation are catalyzed in pocket II.
A phylogenetic analysis of gene sequences, product specificity and structure-function relationships of the maize terpene synthases TPS4 and TPS5 has indicated that the mechanisms of gene duplication and divergence are responsible for the development of diversity in secondary metabolism. We would also like to understand to what extend convergence and repeated evolution are involved to form genes with similar function and expression pattern. In addition to these mechanisms, selection pressure resulting from the function of terpenes in plant defense will be estimated.
The biosynthesis of some terpenes responds to the attack of herbivores whereas others are formed constitutively and restricted to specific maize tissues. The cues for herbivore induction are not only mechanical damage to the plant but also the contact with elicitors that originate from herbivore oral secretions and activate a complex signaling cascade. Similar to the evolution of structural genes, the expression pattern of terpene synthases has been diversified leading to different functions of the terpene products within the plant. Studies of the developmental, spatial and stress-induced gene expression pattern will tell us to what extend gene regulation has diversified and how the genes might have adopted different functions during the phylogeny of the plant.
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