Chemical modification of biomarkers through accelerated degradation: Implications for ancient plant identification in archaeo-organic residues

Biochemical and biomolecular archaeology is increasingly used to elucidate the consumption, use, origin, and trade of plants in the past. However, it can be challenging to use biomarkers to identify the taxonomic origin of archaeological plants due to limited knowledge of molecular survival and degradation for many key plant compounds in archaeological contexts. To gain a fundamental understanding of the chemical alterations associated with chemical degradation processes in ancient samples, we conducted accelerated degradation experiments with essential oil derived from cedar (Cedrus atlantica) exposed to materials commonly found in the archaeological record. Using GC-MS and multivariate analysis, we detected a total of 102 compounds across 19 treatments that were classified into three groups. The first group comprised compounds that were abundant in fresh cedar oil but would be unlikely to remain in ancient residues due to rapid degradation. The second group consisted of compounds that remained relatively stable or increased over time, which could be potential biomarkers for identifying cedar in archaeological residues. Compounds in the third group were absent in fresh cedar oil but were formed during specific experiments that could be indicative for certain storage conditions. These results show that caution is warranted for applying biomolecular profiles of fresh plants to ancient samples and that carefully designed accelerated degradation experiments can, at least in part, overcome this limitation.1. Introduction 2. Experimental Design 3. Results and Discussion 3.1. GC-MS Results and Multivariate Analyses 3.2. Compound Identification 4. Materials and Methods 4.1. Materials 4.2. Sample Preparation, Extraction and Analysis 4.3. Data Pretreatment and Statistical Analysis 5. Conclusion

So much for glucosinolates: A generalist does survive and develop on Brassicas, but at what cost?

While plants produce complex cocktails of chemical defences with different targets and efficacies, the biochemical effects of phytotoxin ingestion are often poorly understood. Here, we examine the physiological and metabolic effects of the ingestion of glucosinolates (GSLs), the frontline chemical defenses of brassicas (crucifers), on the generalist herbivore Helicoverpa armigera. We focus on kale and cabbage, two crops with similar foliar GSL concentrations but strikingly different GSL compositions. We observed that larval growth and development were well correlated with the nutritional properties of the insect diets, with low protein contents appearing to exacerbate the negative effects of GSLs on growth, pupation and adult eclosion, parameters that were all delayed upon exposure to GSLs. The different GSLs were metabolized similarly by the insect, indicating that the costs of detoxification via conjugation to glutathione (GSH) were similar on the two plant diets. Nevertheless, larval GSH contents, as well as some major nutritional markers (larval protein, free amino acids, and fat), were differentially affected by the different GSL profiles in the two crops. Therefore, the interplay between GSL and the nitrogen/sulfur nutritional availability of different brassicas strongly influences the effectiveness of these chemical defenses against this generalist herbivore

Mechanisms of isothiocyanate detoxification in larvae of two belowground herbivores, Delia radicum and D. floralis (Diptera: Anthomyiidae)

International audienceLike aboveground herbivores, belowground herbivores are confronted with multiple plant defense mechanisms including complex chemical cocktails in plant tissue. Roots and shoots of Brassicaceae plants contain the two-component glucosinolate (GSL)-myrosinase defense system. Upon cell damage, for example by herbivore feeding, toxic and pungent isothiocyanates (ITCs) can be formed. Several aboveground-feeding herbivores have developed biochemical adaptation strategies to overcome the GSL-ITC defenses of their host plant. Whether belowground herbivores feeding on Brassica roots possess similar mechanisms has received little attention. Here, we analyze how two related belowground specialist herbivores detoxify the GSL-ITC defenses of their host plants. The larvae of the fly species Delia radicum and D. floralis are common pests and specialized herbivores on the roots of Brassicaceae. We used chemical analyses (HPLC-MS/MS and HPLC-UV) to examine how the GSL-ITC defense system is metabolized by these congeneric larvae. In addition, we screened for candidate genes involved in the detoxification process using RNAseq and qPCR. The chemical analyses yielded glutathione conjugates and amines. This indicates that both species detoxify ITCs using potentially the general mercapturic acid pathway, which is also found in aboveground herbivores, and an ITC-specific hydrolytic pathway previously characterized in microbes. Performance assays confirmed that ITCs negatively affect the survival of both species, in spite of their known specialization to ITC-producing plants and tissues, whereas ITC breakdown products are less toxic. Interestingly, the RNAseq analyses showed that the two congeneric species activate different sets of genes upon ITC exposure, which was supported by qPCR data. Based on our findings, we conclude that these specialist larvae use combinations of general and compound-specific detoxification mechanisms with differing efficacies and substrate preferences. This indicates that combining detoxification mechanisms can be an evolutionarily successful strategy to handle plant defenses in herbivores

Identification of a sulfatase that detoxifies glucosinolates in the phloem-feeding insect Bemisia tabaci and prefers indolic glucosinolates

Cruciferous plants in the order Brassicales defend themselves from herbivory using glucosinolates: sulfur-containing pro-toxic metabolites that are activated by hydrolysis to form compounds, such as isothiocyanates, which are toxic to insects and other organisms. Some herbivores are known to circumvent glucosinolate activation with glucosinolate sulfatases (GSSs), enzymes that convert glucosinolates into inactive desulfoglucosinolates. This strategy is a major glucosinolate detoxification pathway in a phloem-feeding insect, the silverleaf whitefly Bemisia tabaci, a serious agricultural pest of cruciferous vegetables. In this study, we identified and characterized an enzyme responsible for glucosinolate desulfation in the globally distributed B. tabaci species MEAM1. In in vitro assays, this sulfatase showed a clear preference for indolic glucosinolates compared with aliphatic glucosinolates, consistent with the greater representation of desulfated indolic glucosinolates in honeydew. B. tabaci might use this detoxification strategy specifically against indolic glucosinolates since plants may preferentially deploy indolic glucosinolates against phloem-feeding insects. In vivo silencing of the expression of the B. tabaci GSS gene via RNA interference led to lower levels of desulfoglucosinolates in honeydew. Our findings expand the knowledge on the biochemistry of glucosinolate detoxification in phloem-feeding insects and suggest how detoxification pathways might facilitate plant colonization in a generalist herbivore

Activation and detoxification of cassava cyanogenic glucosides by the whitefly Bemisia tabaci

Abstract Two-component plant defenses such as cyanogenic glucosides are produced by many plant species, but phloem-feeding herbivores have long been thought not to activate these defenses due to their mode of feeding, which causes only minimal tissue damage. Here, however, we report that cyanogenic glycoside defenses from cassava (Manihot esculenta), a major staple crop in Africa, are activated during feeding by a pest insect, the whitefly Bemisia tabaci, and the resulting hydrogen cyanide is detoxified by conversion to beta-cyanoalanine. Additionally, B. tabaci was found to utilize two metabolic mechanisms to detoxify cyanogenic glucosides by conversion to non-activatable derivatives. First, the cyanogenic glycoside linamarin was glucosylated 1–4 times in succession in a reaction catalyzed by two B. tabaci glycoside hydrolase family 13 enzymes in vitro utilizing sucrose as a co-substrate. Second, both linamarin and the glucosylated linamarin derivatives were phosphorylated. Both phosphorylation and glucosidation of linamarin render this plant pro-toxin inert to the activating plant enzyme linamarase, and thus these metabolic transformations can be considered pre-emptive detoxification strategies to avoid cyanogenesis