Biosynthesis and Function of Volatile Formation in Woody Plants and Grasses

Dr. Tobias Köllner

Many plants respond to herbivore feeding by producing complex volatile blends. These blends consist of compounds from several chemical classes such as terpenoids, aromatic esters, and green-leaf volatiles. We are investigating the biochemical and genetic basis of herbivore-induced volatile production in woody plants and grasses. Gaining basic knowledge about these processes will help us to elucidate the biological role of single volatile compounds in plant-insect interactions.


The biosynthesis of nitrogen-containing volatiles in poplar

Sandra Irmisch

As a long-lived woody perennial, poplar is exposed to a large variety of biotic stresses throughout its lifetime. Due to the extensive genetic resources available and the simple clonal propagation it is an excellent model for studying the molecular and genetic basis of volatile-mediated defense in a woody plant.

Beside terpenes, nitrogen-containing compounds like benzyl cyanide and amino acid-derived aldoximes represent a major part of the herbivore-induced volatile blend of poplar. However, their biosynthesis as well as their biological relevance are still unknown. In this project we are focusing on P450 enzymes which seem to be involved in the formation of these compounds. Microarray analysis revealed that the members of two P450 gene subfamilies, Cyp79 and Cyp71, are induced after caterpillar feeding. Currently we are working on the heterologous expression and characterization of these P450 enzymes. 

Since Cyp79 and Cyp71 enzymes from other plants are known to be involved in direct defense responses like the production of cyanogenic glucosides and glucosinolates, a comparison of P450 genes involved in direct and indirect defense will help us to study how volatile biosynthetic pathways evolved.   

In a close collaboration with the group of Dr. Sybille Unsicker we will investigate the biological functions of nitrogen-containing volatiles. Knock-out as well as overexpressing poplar trees will be used in EAG and olfactometer experiments to study the role of these compounds in plant-herbivore interactions.


The biosynthesis and biochemical fate of volatile alcohols in Populus trichocarpa

Jan Günther

As a defense against insect herbivores, many plants emit a complex blend of volatile compounds upon herbivory. These volatile blends comprise terpenes, green leaf volatiles, nitrogenous compounds and aromatic compounds. Plant volatiles can reduce the fitness of the herbivore on the emitting plant as they might attract herbivore enemies. Recently we identified several enzymes responsible for the formation of nitrogenous volatiles in poplar. We showed that L-phenylalanine can be converted by CYP79/CYP71 enzymes to benzyl cyanide with phenylacetaldoxime as an intermediate. RNAi-mediated knock-down of the responsible CYP79 genes resulted in trees with reduced emissions of phenylacetaldoxime and benzyl cyanide. Surprisingly, the emission of 2-phenylethanol, a volatile alcohol, was dramatically decreased as well. Feeding of labeled phenylacetaldoxime to poplar leaves resulted in the formation of labeled benzyl cyanide, 2-phenylacetaldehyde and 2-phenylethanol, suggesting a pathway that might connect these herbivore-induced volatiles. We are now characterizing nitrilases, acid and aldehyde reductases, and acyl transferases in aim to understand the enzymatic machinery leading to the formation of volatile aldehydes, alcohols and esters.


The biochemical and genetic basis of terpene formation in poplar

Dr. Tobias Köllner

As other plants, poplar contains a large family of terpene synthase genes (tps) which encode key enzymes of terpene biosynthesis. The recently sequenced genome of Populus trichocarpa revealed 32 full-length tps genes, twelve of which we have already cloned and characterized. The characterization of the other tps genes is currently in progress.

Using a small-scale microarray approach, transcript accumulation of all tps genes will be measured in the different plant organs, under varying biotic and abiotic conditions. The results from these experiments will hint at the function of the enzyme products and help us to understand why and how plants evolved such large terpene synthase gene families.



The evolution of insect-induced terpene biosynthesis in the grasses

Dr. Tobias Köllner

(in collaboration with Dr. Feng Chen, University of Tennessee)

The aim of this project is to investigate the molecular basis of insect-induced terpene biosynthesis in sorghum and the evolution of responsible terpene synthases in different grasses. Currently we identified three terpene synthase genes from sorghum which showed increased expression after herbivore-feeding. Biochemical characterization revealed that the three TPS produce the same sesquiterpenes but with completely different product distributions. Comparative studies with terpene synthase orthologs from sorghum, rice and maize will help us to identify key amino acids which changed during evolution and resulted in altered product specificities.


Identification and characterization of enzymes involved in the biosynthesis and activation of benzoxazinoid derivatives in maize

Vinzenz Handrick

Benzoxazinoids are plant defense compounds which are found mainly in the plants of thegrass family, such as wheat and maize. The biosynthesis of benzoxazinoids (BXDs) in maize has been extensively studied in the last two decades. The pathway starts from indole-3-glycerol phosphate and leads to 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc) and the realted DIBOA-Glc. Maize also produces other BXDs like HDMBOA-Glc, DIM2BOA-Glc, HDM2BOA-Glc, HMBOA-Glc and HBOA-Glc, which are probably derived from DIBOA-Glc and DIMBOA-Glc. However, the biosynthetic sequences leading to this complex mixture of products are currently not known. 

In our attempt to investigate the enzymes responsible for BXD formation in maize, we have started to identify the O-methyltransferase (OMT) which converts DIMBOA-Glc to HDMBOA-Glc (see figure), thought to be more toxic than other BXDs. Recently it was shown that this conversion is strongly upregulated after herbivore feeding. Hence, a detailed understanding of this defense mechanism could speed up the breeding of herbivore-resistant maize lines. In a close collaboration with two research groups in Switzerland we are planning to construct maize lines overexpressing as well as knocked-out in the omt gene. The resistance of these lines against different classes of herbivores will be investigated in laboratory and field experiments.

Beside the formation of BXDs in maize, we are also interested in the activation of these important defense compounds. Activation is based on the formation of highly reactive aglucones f rom the plant glucoside, which occurs after tissue damage through the action of specific β-glucosidases. Two β-glucosidases, Glu1 and Glu2, were already described to be involved in BXD activation. While their biochemical properties were extensively studied in the past, their regulation is poorly understood. BLAST analysis revealed the presence of a small glu1/2-like gene subfamily in the maize genome. Beside glu1 and glu2, it contains five additional glu genes which might also be involved in BXD activation. We will start to analyze the contribution of the single glu genes to aglucone liberation after herbivore feeding.


Ecological functions of maize (Zea mays) volatile sesquiterpenes

Chalie Assefa Fantaye

Plants emit a multitude of constitutive and inducible volatile organic compounds (VOCs) from their tissues. The most abundant and structurally diverse components of these VOCs are the terpenoids. Since plants are part of a complex ecosystem which includes biotic and abiotic factors, they recruit VOCs for their positive and negative interactions within this system. My project focuses on the functional characterization of maize volatile sesquiterpenes, the C15 class of terpenoids, in an ecological context.

Since most of the genes responsible for the production of maize volatile sesquiterpenes are already identified and biochemically characterized in our lab, I used the different terpene synthase genes of maize for the genetic transformation of Arabidopsis thaliana, a plant that does not produce detectable levels of sesquiterpenes at rosette stage. Arabidopsis is then used as a factory to produce individual groups of sesquiterpenes and used as a system to dissect their roles in indirect defense, priming defense, direct anti-fungal and anti-herbivore defenses, and abiotic stress resistance.