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 their natural herbivore predators - indeed, many classes of specialized metabolites are thought to have defensive roles as antifeedants or repellents, including the relatively widespread phenolics, terpenes and alkaloids, as well as the more narrowly distributed glucosinolates and benzoxazinoids, which are the main targets of our current work.
Nevertheless, herbivores manage to use these well-defended plant tissues as food. These successful herbivores are able to do so by employing, among other adaptations, biochemical detoxification strategies that lower or circumvent the toxicity of defensive phytochemicals. These strategies are as varied and complex as the defensive cocktails they can successfully detoxify, but most are still quite poorly understood.
We are applying a variety of chemical and biochemical approaches to elucidate such detoxification reactions and pathways within the herbivores. Isotope labeling and tracing provide us interesting information regarding the quantitative importance of each of these detoxification pathways, and further enzymological studies help us correlate and demonstrate the involvement of each pathway to the effects on the herbivore.
Additionally, we also examine the modes of action of plant defensive compounds within the herbivores, i.e. organism and cellular targets for these phytochemicals which mediate their action as poisons. A systematic comparison of these factors among herbivores of varying degrees of specialization will enable us to better understand the ecological relevance of these biochemical interactions.
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 purpose of this project.
Comparative metabolism of glucosinolates in small herbivores
Plants of the Brassicaceae family produce specific blends of defensive glucosinolates that help give them their corresponding flavors and toxicities. Aliphatic glucosinolates derived from methionine are particularly abundant in broccoli, arugula, cauliflower and Arabidopsis thaliana, where they play a role as phytoanticipins or constitutive defense compounds, especially in tissues/growth stages of high inherent value to plants such as seeds and sprouts. 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 and commercialization of broccoli sprouts as nutraceuticals.
We exploit the native biosynthetic machineries of different plants to generate isotopically labeled glucosinolates (e.g. glucoraphanin) from labeled precursors. These glucosinolates, both in stably (13C)- and radioisotopically (14C)-labeled forms, are proving to be valuable tools for the identification and quantification of the possible detoxification pathways after ingestion. We are now able to examine the biochemical fate of these 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 therefore of critical importance for both plant and predator. We have characterized metabolites excreted by different herbivores after ingestion of glucosinolate-containing Arabidopsis thaliana, 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 putative 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 benefit from the ingestion of these compounds in the same way. Using a proteomic approach, we are investigating the identity and localization of the main protein targets of plant ITCs in insects. Complementing these analyses, we also study the changes in the insect physiology and chemistry, which can give us hints regarding which metabolic functions are altered by ingestion of these compounds. In summary, we want to better understand the mechanisms through which these plant defenses 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. By means of isotopically-labeled BXD administration, we intend to determine the metabolic fate of 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.