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 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.