Guillaume Marti

A specialist root herbivore exploits defensive metabolites to locate nutritious tissues

The most valuable organs of plants are often particularly rich in essential elements, but also very well defended. This creates a dilemma for herbivores that need to maximise energy intake while minimising intoxication. We investigated how the specialist root herbivore Diabrotica virgifera solves this conundrum when feeding on wild and cultivated maize plants. We found that crown roots of maize seedlings were vital for plant development and, in accordance, were rich in nutritious primary metabolites and contained higher amounts of the insecticidal 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and the phenolic compound chlorogenic acid. The generalist herbivores Diabrotica balteata and Spodoptera littoralis were deterred from feeding on crown roots, whereas the specialist D. virgifera preferred and grew best on these tissues. Using a 1,4-benzoxazin-3-one-deficient maize mutant, we found that D. virgifera is resistant to DIMBOA and other 1,4-benzoxazin-3-ones and that it even hijacks these compounds to optimally forage for nutritious roots.

Metabolomics reveals herbivore-induced metabolites of resistance and susceptibility in maize leaves and roots

Plants respond to herbivory by reprogramming their metabolism. Most research in this context has focused on locally induced compounds that function as toxins or feeding deterrents. We developed an ultra-high-pressure liquid chromatography time-of-flight mass spectrometry (UHPLC-TOF-MS)-based metabolomics approach to evaluate local and systemic herbivore-induced changes in maize leaves, sap, roots and root exudates without any prior assumptions about their function. Thirty-two differentially regulated compounds were identified from Spodoptera littoralis-infested maize seedlings and isolated for structure assignment by microflow nuclear magnetic resonance (CapNMR). Nine compounds were quantified by a high throughput direct nano-infusion tandem mass spectrometry/mass spectrometry (MS/MS) method. Leaf infestation led to a marked local increase of 1,3-benzoxazin-4-ones, phospholipids, N-hydroxycinnamoyltyramines, azealic acid and tryptophan. Only few changes were found in the root metabolome, but 1,3-benzoxazin-4-ones increased in the vascular sap and root exudates. The role of N-hydroxycinnamoyltyramines in plant-herbivore interactions is unknown, and we therefore tested the effect of the dominating p-coumaroyltyramine on S. littoralis. Unexpectedly, p-coumaroyltyramine was metabolized by the larvae and increased larval growth, possibly by providing additional nitrogen to the insect. Taken together, this study illustrates that herbivore attack leads to the induction of metabolites that can have contrasting effects on herbivore resistance in the leaves and roots.

Differentiation of lemon essential oil based on volatile and non-volatile fractions with various analytical techniques: a metabolomic approach

Due to the importance of citrus lemon oil for the industry, fast and reliable analytical methods that allow the authentication and/or classification of such oil, using the origin of production or extraction process, are necessary. To evaluate the potential of volatile and non-volatile fractions for classification purposes, volatile compounds of cold-pressed lemon oils were analyzed, using GC-FID/MS and FT-MIR, while the non-volatile residues were studied, using FT-MIR, (1)H-NMR and UHPLC-TOF-MS. 64 Lemon oil samples from Argentina, Spain and Italy were considered. Unsupervised and supervised multivariate analyses were sequentially performed on various data blocks obtained by the above techniques. Successful data treatments led to statistically significant models that discriminated and classified cold-pressed lemon oils according to their geographic origin, as well as their production processes. Studying the loadings allowed highlighting of important classes of discriminant variables that corresponded to putative or identified chemical functions and compounds.

Transcriptome diversity among rice root types during asymbiosis and interaction with arbuscular mycorrhizal fungi

Significance Plant roots function as an interface between plants and the complex soil environment. Root systems of higher plants consist of different root types (RTs) that maximize their adaptive potential in heterogenous soil for nutrient uptake and anchorage. This study pioneers the molecular examination of individual RTs of adult rice root systems. The global signature of the transcriptional activity of each RT reveals significant quantitative and qualitative differences that predict functional diversity and specialization. Interaction with naturally prevalent beneficial mycorrhizal fungi profoundly modulated the relationship across the RTs such that the crown root transcriptome resembled that of lateral roots. The alteration of secondary cell wall synthesis in colonized roots is consistent with previously reported changes in root system architecture of mycorrhizal plants. Root systems consist of different root types (RTs) with distinct developmental and functional characteristics. RTs may be individually reprogrammed in response to their microenvironment to maximize adaptive plasticity. Molecular understanding of such specific remodeling—although crucial for crop improvement—is limited. Here, RT-specific transcriptomes of adult rice crown, large and fine lateral roots were assessed, revealing molecular evidence for functional diversity among individual RTs. Of the three rice RTs, crown roots displayed a significant enrichment of transcripts associated with phytohormones and secondary cell wall (SCW) metabolism, whereas lateral RTs showed a greater accumulation of transcripts related to mineral transport. In nature, arbuscular mycorrhizal (AM) symbiosis represents the default state of most root systems and is known to modify root system architecture. Rice RTs become heterogeneously colonized by AM fungi, with large laterals preferentially entering into the association. However, RT-specific transcriptional responses to AM symbiosis were quantitatively most pronounced for crown roots despite their modest physical engagement in the interaction. Furthermore, colonized crown roots adopted an expression profile more related to mycorrhizal large lateral than to noncolonized crown roots, suggesting a fundamental reprogramming of crown root character. Among these changes, a significant reduction in SCW transcripts was observed that was correlated with an alteration of SCW composition as determined by mass spectrometry. The combined change in SCW, hormone- and transport-related transcript profiles across the RTs indicates a previously overlooked switch of functional relationships among RTs during AM symbiosis, with a potential impact on root system architecture and functioning.

Study of Leaf Metabolome Modifications Induced by UV-C Radiations in Representative Vitis, Cissus and Cannabis Species by LC-MS Based Metabolomics and Antioxidant Assays

UV-C radiation is known to induce metabolic modifications in plants, particularly to secondary metabolite biosynthesis. To assess these modifications from a global and untargeted perspective, the effects of the UV-C radiation of the leaves of three different model plant species, Cissus antarctica Vent. (Vitaceae), Vitis vinifera L. (Vitaceae) and Cannabis sativa L. (Cannabaceae), were evaluated by an LC-HRMS-based metabolomic approach. The approach enabled the detection of significant metabolite modifications in the three species studied. For all species, clear modifications of phenylpropanoid metabolism were detected that led to an increased level of stilbene derivatives. Interestingly, resveratrol and piceid levels were strongly induced by the UV-C treatment of C. antarctica leaves. In contrast, both flavonoids and stilbene polymers were upregulated in UV-C-treated Vitis leaves. In Cannabis, important changes in cinnamic acid amides and stilbene-related compounds were also detected. Overall, our results highlighted phytoalexin induction upon UV-C radiation. To evaluate whether UV-C stress radiation could enhance the biosynthesis of bioactive compounds, the antioxidant activity of extracts from control and UV-C-treated leaves was measured. The results showed increased antioxidant activity in UV-C-treated V. vinifera extracts.

Comprehensive profiling and marker identification in non-volatile citrus oil residues by mass spectrometry and nuclear magnetic resonance

The detailed characterization of cold-pressed lemon oils (CPLOs) is of great importance for the flavor and fragrance (F&F) industry. Since a control of authenticity by standard analytical techniques can be bypassed using elaborated adulterated oils to pretend a higher quality, a combination of advanced orthogonal methods has been developed. The present study describes a combined metabolomic approach based on UHPLC-TOF-MS profiling and (1)H NMR fingerprinting to highlight metabolite differences on a set of representative samples used in the F&F industry. A new protocol was set up and adapted to the use of CPLO residues. Multivariate analysis based on both fingerprinting methods showed significant chemical variations between Argentinian and Italian samples. Discriminating markers identified in mixtures belong to furocoumarins, flavonoids, terpenoids and fatty acids. Quantitative NMR revealed low citropten and high bergamottin content in Italian samples. The developed metabolomic approach applied to CPLO residues gives some new perspectives for authenticity assessment.

LC-MS/MS quantitative determination of Tetrapterys mucronata alkaloids, a plant occasionally used in ayahuasca preparation.

INTRODUCTION Tetrapterys mucronata Cav. (Malpighiaceae) is a plant used in some regions of Brazil in the preparation of ayahuasca. OBJECTIVE To determine the content of the main tryptamine alkaloids in the stem bark of T. mucronata Cav. and assess their possible toxic and hallucinogenic properties based on the doses found in a water decoction that mimics the ayahuasca preparation. METHODS Four alkaloids previously described for their toxic and hallucinogenic properties were quantitated by multiple reaction monitoring HPLC combined with electrospray ionisation and tandem MS (HPLC-ESI/MS/MS) in the water decoction and ethanolic extracts from the bark of T. mucronata. RESULTS Exhaustive extraction of the stem barks with ethanol revealed the following alkaloid levels: bufotenine (1) 3.26 ± 0.31 mg/g, 5-methoxy-N-methyltryptamine (2) 0.88 ± 0.08 mg/g, 5-methoxy-bufotenine (3) 3.07 ± 0.22 mg/g and 2-methyl-6-methoxy-1,2,3,4-tetrahydro-β-carboline (4) 0.14 ± 0.004 mg/g. The water decoction presented slightly lower levels, ranging between 2.32 ± 0.14, 0.50 ± 0.04, 1.53 ± 0.09 and 0.10 ± 0.01 mg/g for (1), (2), (3) and (4) respectively. CONCLUSIONS The HPLC-ESI/MS/MS quantitation revealed significant alkaloid levels, in particular for bufotenine and 5-methoxy-bufotenine. As such compounds are known for their toxic and hallucinogenic properties, these results indicate that the consumption of this plant as an ingredient in ayahuasca preparations may present a risk to consumers.

Cucurbitacins from the Leaves of Citrullus colocynthis (L.) Schrad

Two new tetracyclic cucurbitane-type triterpene glycosides were isolated from an ethyl acetate extract of Citrullus colocynthis leaves together with four known cucurbitacins. Their structures were established on the basis of their spectroscopic data (mainly NMR and mass spectrometry). Evaluation of the in vitro cytotoxic activity of the isolated compounds against two human colon cancer cell lines (HT29 and Caco-2) and one normal rat intestine epithelial cell line (IEC6), revealed that one of the isolated compounds presented interesting specific cytotoxic activity towards colorectal cell lines.

Stilbenes: Biomarkers of Grapevine Resistance to Disease of High Relevance for Agronomy, Oenology and Human Health

Stilbenic phytoalexins are key defence molecules implicated in the resistance of grapevine cultivars to three major fungal pathogens, Botrytis cinerea (grey mould of grape), Plamopara viticola (downy mildew) and Erysiphe necator (powdery mildew). HPLC analysis of stilbenes is an efficient method to evaluate the ability of the vine plants to inhibit the development of fungal pathogens. Resistant grapevine varieties react very rapidly to infections by producing high concentrations of the most toxic stilbenes, δ-viniferin and pterostilbene, at the sites of infection. Monitoring of such stress biomarkers is also of great interest for evaluating the efficiency of priming molecules at inducing the grapevines’ natural defence responses. In addition, these compounds have various beneficial effects on human health, acting as anti-oxidants and also as potential chemopreventive agents. The diversity of stilbenes is intriguing, and new holistic analytical approaches, such as metabolomics, that are widely used for wine classification also have great potential for the comprehensive study of responses of Vitaceae to biotic and abiotic stress.

Search for Low-Molecular-Weight Biomarkers in Plant Tissues and Seeds Using Metabolomics: Tools, Strategies, and Applications

This chapter summarises the metabolomic strategies currently in force used in plant science and describes the methods used. The metabolite profiling and fingerprinting of plant tissues through MS- and/or NMR-based approaches and the subsequent identification of biomarkers is detailed. Strategies for the microisolation and de novo identification of unknown biomarkers are also discussed. The various approaches are illustrated by a metabolomic study of the maize response to herbivory. A review of recent metabolomic studies performed on seed and crop plant tissues involving various analytical strategies is provided.