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 poplar

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 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.

 

Exploring the underground: How poplar roots defend against herbivores

Nathalie Lackus

In response to herbivory, many plants produce defense compounds which can either be toxic or repellant for the herbivore or attractive for natural enemies of the herbivore. These direct and indirect defense mechanisms often present strong barriers against various attackers. Such induced plant defense mechanisms have been intensively studied above-ground, but little is known about the induced defenses of roots, especially those of trees. In this regard, we investigate the herbivory-induced accumulation of chemical metabolites in roots of the Western balsam poplar (Populus trichocarpa). Feeding of cockchafer (Melolontha melolontha) larvae on poplar roots leads to a highly increased accumulation of salicylaldehyde. To investigate the biochemical mechanisms of the observed herbivory-induced root response, a transcriptome dataset was generated to identify all differentially expressed genes in poplar roots. The further aim of the study is the identification and characterization of enzymes which are responsible for the formation of salicylaldehyde. Putative candidate genes of a ß-oxidative biosynthetic pathway were identified and we are now characterizing the cinnamic acid-CoA ligases (CNL), cinnamoyl-CoA hydratases/dehydrogenases (CHD) and 3-ketoacyl-CoA-thiolases (KAT) in poplar. Further on, transgenic trees will be generated and then used in behavioral assays with cockchafer larvae in order to elucidate the biological role of salicylaldehyde as defense compund.

The odour of roots: Biochemical basis of terpene biosynthesis in poplar roots

Nathalie Lackus

Plants produce and emit a large variety of volatile organic compounds that belong to several chemical classes as for instance terpenoids or green leaf volatiles. Preliminary experiments revealed that feeding of cockchafer (Melolontha melolontha) larvae on poplar roots induces the emission of volatile monoterpenes. As other plants, poplar contains a large family of terpene synthase genes (tps) which encode key enzymes of terpene biosynthesis. The aim of the study is the identification of terpene synthases responsible for the formation of volatile monoterpenes in poplar roots. Identified candidate genes will be further characterized and their enzymatic activity will be examined in vitro. Further on, amino acids in the active site of the terpene synthases will be specifically changed to alter the product formation of the terpene synthases.

Biochemical and functional aspects of the metabolism of DIMBOA-Glc in maize and wheat

Christiane Förster

Benzoxazinoids (BXDs) are plant secondary metabolites that are well known for their defensive and allelopathic properties. They are mainly produced in plants of the grass family (Poaceae), including agricultural crops such as maize, wheat, and rye. The biosynthesis of BXDs has been extensively studied in maize and especially the biosynthetic pathway for the benzoxazinoid hydroxamic acids is fully elucidated in this plant. The core pathway starts from indole-3-glycerol phosphate, which undergoes several oxidations, a glucosylation, and a methylation leading to 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc). DIMBOA-Glc is the most abundant BXD in undamaged maize, however, after herbivore feeding it is converted to a variety of other compounds such as HDMBOA-Glc, DIM2BOA-Glc , and HDM2BOA-Glc. In collaboration with the groups of Georg Jander (Boyce Thompson Institute, Ithaca, USA) and Matthias Erb (University of Bern, Switzerland), we have recently identified and characterized the enzymes involved in the formation of HDMBOA-Glc, DIM2BOA-Glc, and HDM2BOA-Glc in maize (see figure). The aim of the present study is to identify respective enzyme homologues in wheat and to investigate how they might have evolved. Preliminary BLAST analysis with the wheat genome revealed no direct orthologues, suggesting a convergent evolution of DIMBOA-Glc metabolism in the grasses. Since fungus infestation can induce the conversion of DIMBOA-Glc into other BXDs in wheat, a transcriptomic dataset derived from non-infested and fungus-infested wheat plants will help us to identify upregulated candidate genes. Further on, we are interested in elucidating the biosynthesis of benzoxazinoid lactams such as HMBOA-Glc in both maize and wheat. These compounds are frequently found in the leaves besides benzoxazinoid hydroxamid acids, but the enzymes responsible for their formation are still unknown and the biological roles of these compounds are unclear.

The role of beta-glucosidases and beta-glucosidase-aggregating factors in the activation of benzoxazinoids in maize roots

Diana Radisch

β-Glucosidases are found in all domains of living organisms, where they play essential roles in the removal of nonreducing terminal glucosyl residues from saccharides and glycosides. Plant β-glucosidases are involved in many physiological processes like cell wall formation, phytohormone release and activation of defense compounds. Activation of secondary metabolites by de-glycosylation is a widespread anti-herbivore defense strategy which allows plants to store harmless glycosides and hydrolyze them to toxic products upon attack. The main insect resistance factors in maize plants are 1,4-benzoxazin-3-one derivates (BXDs), like DIMBOA-Glc, which are stored as glucosides and activated by plant β-glucosidases. Two β-glucosidases (ZmGlu1 & ZmGlu2) have been shown to hydrolyze DIMBOA-Glc and DIBOA-Glu. There are 26 GH1 genes found in the genome of the maize inbred line B73. But only 6 other ZmGlu genes (ZmGlu3-8) form a distinct gene subfamily together with ZmGlu1 and ZmGlu2. Since ZmGlu3-8 share 70 – 80% similarity with ZmGlu1 & 2, it is likely that they encode enzymes with similar catalytic properties that might also accept benzoxazinoids as substrates.
In this project I am going to characterize the expression levels of ZmGlu1-8 in different maize organs and after different biotic stresses. Furthermore the substrat specifities of the recombinant enzymes will be determined in assays containing different BXDs and artificial substrates.
Some maize lines (“null” lines) possess only marginal soluble β-glucosidase activity after tissue disruption. In these lines, β-glucosidases are bound to a protein called β-glucosidase-aggregating factor (BGAF) and form insoluble protein complexes. BGAF-β-glucosidase aggregation has been suggested to protect β-glucosidases from proteolytic degradation in the insect gut, however, the exact function of BGAFs, even in the context of β-glucosidase stabilization, is yet unclear. The interactions of purified recombinant BGAF proteins with recombinant ZmGlu proteins will be tested with pull-down and gel shift assays. Furthermore, I will investigate the cellular localization of ZmGlu proteins and BGAFs in crown roots by using specific antibodies.