Detoxification and Mode of Action of Plant Compounds in Herbivores
In our research group we want to better understand the biochemistry of the interactions between herbivores and the plant chemical defenses they encounter while feeding. Plants use cocktails of defensive chemicals for protection against herbivore predators - indeed, many specialized metabolites are thought to have defensive roles as antifeedants or repellents. These include the relatively widespread phenolics, terpenes and alkaloids, as well as the more narrowly distributed glucosinolates, cyanogenic glucosides and benzoxazinoids, which are the main targets of our current work. Nevertheless, herbivores manage to use these well-defended plant tissues as food, and some crop pests even compete with humans for food sources. These successful herbivores are able to do so by employing, among other adaptations, detoxification strategies that lower or circumvent the toxicity of defensive phytochemicals. The chemistry and biochemistry behind these strategies are as varied and complex as the plant defensive cocktails, but are still quite poorly understood even in economically important crop-pest interactions. On the other hand, the molecular mechanisms through which plant chemical defenses exert their toxic effects are also in most cases still not identified. While some generalized modes of action have been proposed, the particular targets and the metabolic ramifications of intoxication have been largely ignored.
We are applying a variety of chemical and biochemical approaches to tackle these two aspects of plant-pest interactions. Our goals are to identify modes of action of plant defenses, as well as to elucidate and quantify corresponding detoxification pathways within the herbivores. Isotope labeling and tracing provide us crucial quantitative information on the importance of each detoxification pathway, and further enzymological and silencing/knock-down studies allow us to demonstrate the involvement of each pathway, and how they may benefit the herbivore. Proteomic analyses and assays in cultured cells help further clarify the processes directly and indirectly affected by plant-derived toxins.
Our current projects can be divided, based in the host-plant chemistry, in:
Cabbage, broccoli, radish, wasabi, and even the model plant Arabidopsis thaliana… all of these plants and others in the order Brassicales produce glucosinolates, the nitrogen- and sulfur-containing components of the so-called “mustard oil bomb”, an uncommon defense tactic against herbivores. Upon chewing-induced “detonation” of this bomb, a cascade of enzymatic chemical reactions takes place. Together, these transform otherwise harmless plant chemicals (glucosinolates) into potent feeding deterrents and even poisons, namely isothiocyanates, nitriles, and thiocyanates, among others. When humans bite into these plants, they experience a pleasant sharp flavor originating from glucosinolate hydrolysis products, whereas for smaller herbivores this bite can be toxic or deadly. Some herbivores, however, have biochemical means that enable them to exploit these plants as food sources. Elucidating the adaptive biochemical strategies of herbivores with respect to glucosinolates and the mechanisms of toxicity of these compounds are the main purposes of this project.
Comparative metabolism of glucosinolates in small herbivores
Plants of the Brassicaceae family produce blends of defensive glucosinolates that help to protect them from herbivores and that also give them their corresponding flavors. Aliphatic glucosinolates derived from methionine are particularly abundant in broccoli, arugula, cauliflower and Arabidopsis thaliana. Glucoraphanin (4-methylsulfinylbutyl glucosinolate), one such aliphatic glucosinolate, has been receiving increasing amounts of interest for the anti-cancer activities of its derived isothiocyanate (sulforaphane), which have led to the marketing of broccoli sprouts as nutraceuticals. Benzenic glucosinolates such as sinalbin (4-hydroxybenzyl glucosinolate) and indolic glucosinolates such as glucobrassicin (indol-3-yl glucosinolate), on the other hand, have very different side-chain chemistries and reactivities, and undergo other detoxification reactions after ingestion. We exploit the native biosynthetic machineries of different crucifer plants to generate isotopically labeled glucosinolates from labeled amino acid precursors. These glucosinolates, both in stably (13C)- and radioisotopically (14C)-labeled forms, are valuable tools for the identification and quantification of detoxification pathways after ingestion. We are now able to examine the biochemical fate of these diverse glucosinolates during digestion and within the herbivores’ organisms, especially the localization, sequence and variety of reactions that take place after their activation by myrosinases. Such detoxification pathways contribute to the eventual success or avoidance of herbivory, and are of critical importance for both plant and predator. We have characterized metabolites excreted by different herbivores after ingestion of glucosinolates, with these including isothiocyanates conjugated to amino acids and to glutathione. We are now further determining the relative importance of these metabolic pathways in different herbivores, and better characterizing the enzymatic activities that lead to their formation.
Modes of action of isothiocyanates in insects
Although a portion of the glucosinolates ingested by generalist insect herbivores are metabolized (e.g. via conjugation), a large amount of isothiocyanate (ITC) is still excreted unmodified. This free ITC pool leads to negative growth effects, delayed development and even the insect’s death. The aim of this project is to understand the biological targets of ITCs in the insect organism, and the effects of these compounds in the insect’s metabolism. Previous studies in mammal models have succeeded in identifying some of the proteins that mediate the biological activities of ITCs, e.g. in humans, where they can present anti-inflammatory and anti-cancer properties. However, insect herbivores don’t see such benefits after the ingestion of these compounds. We have been studying the changes in the insect physiology and chemistry caused by ITCs, which can give us hints regarding which metabolic functions are altered by ingestion of these compounds. We have determined that ingestion of ITC leads to drastic changes in levels of glutathione (GSH), as well as its precursor amino acid cysteine, with ramifications in protein and fat metabolism. Complementing these analyses, we are also investigating the identity and localization of the main protein targets of ITCs in insects using a proteomic approach. In summary, our results will give us an understanding of the mechanisms through which glucosinolates exert their toxic activities to successfully deter herbivory.
Benzoxazinoids (BXDs) are tryptophan-derived defense compounds present in a small number of agriculturally relevant grass (Poaceae) crops, including maize, wheat and rye. As is the case with glucosinolates, these compounds are also stored in the intact plants as relatively stable glucosides. Physical disruption of the tissue and the activity of glucosidases lead to formation of unstable aglucones that mediate the toxicity of this class of compounds. Surprisingly, however, some insects can cope with the presence of these compounds in their food, and can consume BXD-defended plants undeterred. The aim of this project is to investigate the detoxification strategies used by lepidopterans to avoid the toxicity of benzoxazinoids. We have found that some Spodoptera species, including the maize pest S. frugiperda (fall armyworm), catalyze the reglucosylation of benzoxazinoid aglucones with the inversion of a chiral center relative to the original plant glucoside. The resulting product (2S)-DIMBOA-Glc cannot be re-activated by the plant, thus losing its defensive function. The enzyme(s) responsible for this conversion are currently being investigated. By means of isotopically-labeled BXD administration, we intend to further determine and quantify the metabolic fate of other individual BXDs in the insect’s organism, which includes the characterization of resulting metabolites, the determination of different detoxification routes used by the insects and their relative importance in vivo. Based on the main detoxification strategies found in insects, we will further study the enzymes involved in BXD detoxification pathways by both molecular biological and classical biochemical approaches, evaluating their relevance in vivo and correlating their activities with the capacity of different herbivores to tolerate BXDs.
This project is being performed in collaboration with Dr. Matthias Erb and laboratories in Switzerland as part of a Sinergia initiative.
Cyanogenic glucosides (CNGlcs) are present as an activated chemical defense in many agriculturally relevant plants, such as the grass crops oats and maize, fruit crops such as apples and mangoes, and in the South American and sub-Saharan staple food cassava. Removal of a CNGlc sugar group leads to release of HCN, which inhibits cellular respiration leading to toxicity and death. We want to understand how detoxification of CNGlcs and other cassava defensive chemicals allows some sub-Saharan Bemisia tabaci whitefly strains to successfully and super-abundantly colonize cassava. Colonization by these phloem feeders leads to large losses in cassava production, either directly or via B. tabaci-vectored viruses.
This project is being performed in collaboration with Prof. Shai Morin and Dr. Osnat Malka from the Hebrew University of Jerusalem as part of a Bill & Melinda Gates Foundation-funded initiative led by the Natural Resources Institute of the University of Greenwich.
Past group members
Dr. Naveen Chandra Bisht (2013, 2014, 2015, visiting scientist, Max Planck India Fellow), Roshan Kumar (2015, visiting student) – National Institute of Plant Genome Research, New Delhi
Ross Ward (2015, Intern) – Bachelor student, UC Berkeley
Birthe Förster (2015, Intern)
Nils Meyer (2014, M.Sc. Student) – Doctoral student, Friedrich-Schiller-Universität Jena
Ana Maria de Melo Moreira Gomes (2013/2014, Intern)
Anja Theumer (2014, HiWi)
Susann Rudolph (2013/2014, HiWi)
Thies Gülck (2013/2014, HiWi) – M.Sc. student, University of Copenhagen
Dr. Katharina Schramm (2004-2013, PhD, post-doc) – Post-doc fellow at the University of Utah
Cady Pearce (2013, Intern) – B.Sc. student, Brown University
Blair Blanchette (2013, Intern) – Undegraduate student, University of Virginia (finished), Grassroots Coordinator for the Chesapeake Bay Foundation
Emily Kearney (2012/2013, Intern) – Doctoral student, UC Berkeley
Javier Garcia Jorge (2012/2013, Intern)
Bastian Kindermann (2012/2013, HiWi)
Michael Thieme (2011/2012, Intern) – Doctoral student, Universität Basel
Isabel Meininger (2010-2011, HiWi)