Grapevine trunk diseases (GTDs), such as esca, pose a major threat to viticulture worldwide and are associated with complex biochemical responses in woody tissues. Comprehensive metabolome coverage remains a challenge, as conventional methods often overlook non-polar metabolites critical to plant defense mechanisms. This study aimed to expand metabolome and lipidome coverage of grapevine wood by integrating complementary LC-MS approaches, in order to identify metabolic signatures linked to pathogenic fungi and to a biocontrol agent. Woody tissues of Vitis vinifera cv. Cabernet-Sauvignon were inoculated with Phaeomoniella chlamydospora, Phaeoacremonium minimum, and/or the biocontrol fungus Trichoderma atroviride (Vintec®). A biphasic extraction was coupled with three orthogonal LC-MS methods—reverse-phase (RP), hydrophilic interaction chromatography (HILIC), and lipidomics-focused RP. Data were processed through the MSCleanR workflow and integrated using the DIABLO multi-block statistical framework. Compound classification was performed with NPClassifier. The multiplexed strategy enabled the annotation of 1,425 unique features, representing an 83

Gaultheria procumbens essential oil (GEO) is a natural source of defense inducers, as it is essentially composed of methyl salicylate (MeSA), a compound that can be converted in plant tissues into salicylic acid (SA), a central phytohormone regulating plant immunity. To gain insight into the response of plants to GEO treatment, we performed transcriptomic and metabolomic analyses of GEO-treated Arabidopsis thaliana plants. RNA sequencing analysis showed that fewer than 300 genes were differentially regulated, most of them belonging to functional categories related to receptors, signaling pathways, and transcription factors associated with plant defense or SA biosynthesis and signal transduction. Using the Arabidopsis SA biosynthesis-deficient sid2 line, we investigated MeSA metabolism in plant leaves following foliar GEO treatment. Quantification of MeSA, free SA, and total SA revealed that MeSA was mainly metabolized within 48 h after treatment and that GEO treatment led to the accumulation of conjugated forms of SA. Next, an untargeted metabolomic study was performed on GEO-treated leaves and on plants treated with the SA analog acibenzolar-S-methyl (Bion). This analysis revealed the accumulation of several benzoic acid derivatives upon GEO treatment but no accumulation of defense-related compounds such as indoles. By contrast, Bion treatment induced several indolic derivatives, including indole-3-carboxylic acid. This suggests that Bion directly triggered defense responses, whereas GEO treatment exerted a priming effect. Accordingly, metabolomic responses of A. thaliana leaves to Colletotrichum higginsianum infection showed a stronger accumulation of plant defense metabolites in GEO-treated leaves compared with untreated leaves.Copyright (c) 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Confident metabolite annotation remains a critical bottleneck in untargeted LC-MS metabolomics, with experimental spectral libraries covering only 5-20% of detected features. While in silico tools generate extensive candidate lists per feature, top-ranked predictions frequently fail to reflect true molecular identities, leading to high false annotation rates. We present multi-similarity Network-based annotation (MS-Net), an accessible workflow that integrates mass spectral similarity networks, molecular structure similarity (Tanimoto metrics), and taxonomic knowledge to prioritize annotations within vast candidate spaces. High-confidence annotations from authentic standards, spectral libraries, and taxonomically filtered candidates seed iterative propagation throughout mass spectral similarity networks. The workflow employs a composite Link Score combining structural, spectral, and computational evidence to rescue correct annotations from lower-ranked positions. Applied to Cannabis sativa extracts (2595 features to 1297 after filtering), MS-Net assigned 1275 compounds from an initial candidate space of over 118,000 structures. Notably, 53% of final annotations were rescued from ranks 2-50, demonstrating correction of initial in silico ranking. The workflow successfully reconstructed known cannabinoid biosynthetic pathways, validating biological coherence. MS-Net is freely available as a KNIME workflow with complete documentation at https://forge.inrae.fr/metatoul/equipe-agromix/ms-net, enabling reproducible, offline annotation suitable for systems biology integration.

Heterophylly, characterized by distinct leaf morphologies, is an adaptive strategy evolved by some aquatic plants to cope with the contrasting environments in which they grow. It is shaped by environmental factors and by hormones, particularly abscisic and gibberellic acids. We conducted a comparative multi-omics analysis of submerged and floating leaves of Potamogeton nodosus, a heterophyllous species of the Alismatales order. Genome sequencing has confirmed its tetraploid status. A phylogenomic analysis has identified genes associated with aquatic adaptation, notably genes possibly involved in aurone biosynthesis, which are antioxidant flavonoids. Transcriptomic and metabolomic analyses have revealed distinct gene expression and metabolite patterns depending on the leaf position in the water column. The floating leaves exhibited upregulated genes associated with photosynthetic and metabolic activities, and with the accumulation of UV-protective metabolites reflecting light capture and photoprotection optimization. Conversely, submerged leaves showed upregulated genes associated with homeostatic processes, defense responses, and cell wall remodeling, reflecting tissue maintenance. We have also identified genes involved in the abscisic and gibberellic acids pathways. The developmental analyses highlighted active growth processes in young leaves. This multi-omic study provides an integrated understanding of the complex molecular mechanisms underlying the adaptation of P. nodosus to a dual lifestyle.

Gousiekte ("quick disease") is a fatal plant toxicosis affecting livestock in South Africa. Gousiekte is characterized by the sudden death of animals following the ingestion of leaves of several species of Rubiaceae, most notably Vangueria pygmaea , Pavetta harborii , P. schumanniana and Fadogia homblei . The toxin causing gousiekte is pavettamine, a hydroxylated polyamine pavettamine for which no biosynthetic pathway is known. Interestingly, all plants known to contain pavettamine also feature obligate endophytes of the Burkholderiaceae family, in particular belonging to the Caballeronia and Paraburkholderia genera. We identified a cluster of three genes conserved in all Burkholderia s.l. endophytic symbionts of plants containing pavettamine. Constitutive expression the pavABC gene cluster in a strain of Paraburkholderia caledonica isolated from leaves of Fadogia homblei resulted in detectable levels of pavettamine in cultures, and targeted gene deletions showed the involvement of all three genes in its biosynthesis. Genomes of important animal and human pathogens of the Burkholderia pseudomallei complex encode functional homologs of the pavABC genes, indicating a potentially unrecognized role of pavettamine in disease or complications from infections.

Many flaviviruses with high pandemic potential are transmitted through mosquito bites. While mosquito saliva is essential for transmission and represents a promising pan-flaviviral target, there is a dearth of knowledge on salivary metabolic transmission enhancers. Here, we show that extracellular vesicle (EV)-derived sphingomyelins in mosquito saliva reconfigure the human cell lipidome to increase viral protein levels, boosting skin infection and enhancing transmission for flaviviruses. Lipids within internalized mosquito EVs enhance infection in fibroblast and immune human primary cells for multiple flaviviruses. Mosquito EV lipids selectively increase viral translation by inhibiting infection-induced endoplasmic reticulum (ER)-associated degradation of viral proteins. Infection enhancement solely results from the sphingomyelins within salivary mosquito EVs that augment human cell sphingomyelin concentration. Finally, EV-lipid co-inoculation exacerbates disease severity in vivo in mouse transmission assays. By discovering and elucidating how metabolic components of mosquito saliva promote transmission of flaviviruses, our study unveils lipids as a new category of targets against vectored transmission.

The sweetness of Stevia rebaudiana (Bertoni) leaves is attributed to steviol glycosides (SGs). A method to analyze more extensively and decipher the structures of SGs is needed to understand (i) the biochemical determinism of SG diversity and (ii) the variability of their sensorial attributes. SGs from 20 genotypes of S. rebaudiana (Bertoni) were extracted via liquid extraction and tentatively identified by using LC-ESI-HRMS untargeted metabolomics, enabling the annotation of 114 SGs at different confidence levels. We identified the presence of original malonylated-SGs (up to 36%), acetylated-SGs, and other unidentified families, and confirmed the structure of malonyl-Reb A through 1D and 2D-NMR analyses after purification. Targeted identification and absolute quantification of 16 SGs were performed using analytical standards. Statistical analyses were applied to SG-based classification of stevia within 5 classes. This approach, combining targeted and untargeted metabolomics, may facilitate genotype selection for breeding programs and industrial applications based on SG proportions.

This chapter explores advances and methodologies in high-throughput metabolic phenotyping through metabolomics and predictive modeling to enhance the understanding of plant metabolism. Key techniques, data analysis tools, and applications in plant science research are discussed. The potential of predictive modeling to identify new metabolic pathways and markers associated with plant performance and improve crop traits is highlighted. Future directions and challenges in the field are also examined.

Metabolomics, the comprehensive analysis of low-molecular-weight metabolites (typically below 1500 DA) in biological systems, relies heavily on mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. Each technique has inherent strengths and weaknesses. MS offers high sensitivity and is commonly coupled with chromatography to analyze complex matrices, yet it is destructive, has limited reproducibility, and provides limited structural information. NMR, while less sensitive, is non-destructive and enables structural elucidation and precise quantification. Recent studies increasingly employ data fusion (DF) strategies to combine the complementary information from NMR and MS, aiming to enhance metabolomic analyses. This review summarizes DF methodologies using NMR and MS data in metabolomics studies over the past decade. A comprehensive search of SciFinder, Scopus, and Clarivate Web of Science databases was conducted to analyze fusion techniques, methods, and statistical models. The review emphasizes the growing importance of DF in metabolomics, showing its capacity to provide a more comprehensive view of biochemical processes across diverse biological systems, including clinical, plant, and food matrices.

Annotation is the process of assigning features in mass spectrometry metabolomics data sets to putative chemical structures or "analytes." The purpose of this study was to identify challenges in the annotation of untargeted mass spectrometry metabolomics datasets and suggest strategies to overcome them. Toward this goal, we analyzed an extract of the plant ashwagandha (Withania somnifera) using liquid chromatography-mass spectrometry on two different platforms (an Orbitrap and Q-ToF) with various acquisition modes. The resulting 12 datasets were shared with ten teams that had established expertise in metabolomics data interpretation. Each team annotated at least one positive ion dataset using their own approaches. Eight teams selected the positive ion mode data-dependent acquisition (DDA) data collected on the Orbitrap platform, so the results reported for that dataset were chosen for an in-depth comparison. We compiled and cross-checked the annotations of this dataset from each laboratory to arrive at a "consensus annotation," which included 142 putative analytes, of which 13 were confirmed by comparison with standards. Each team only reported a subset (24 to 57%) of the analytes in the consensus list. Correct assignment of ion species (clusters and fragments) in MS spectra was a major bottleneck. In many cases, in-source redundant features were mistakenly considered to be independent analytes, causing annotation errors and resulting in overestimation of sample complexity. Our results suggest that better tools/approaches are needed to effectively assign feature identity, group related mass features, and query published spectral and taxonomic data when assigning putative analyte structures.

Some plants engage in permanent, vertically transmitted symbioses with bacteria. Often, these bacteria are hosted extracellularly within structures on the leaves, where they produce specialized bioactive metabolites that benefit their host. These associations are highly specific, with one plant species associating with a single bacterial species, but little is known about how these symbioses originate and how specificity is maintained. In this study, we show that the symbiotic association between a wild yam and a bacterium can be manipulated experimentally and that bacteria-free plants are open to colonization by environmental bacteria. Through metabolic profiling, we show that the endophytic niche is rich in organic acids and intermediates of the tricarboxylic acid cycle cycle. Environmental bacteria capable of utilizing these acids, such as the soil bacterium Pseudomonas putida, readily colonize aposymbiotic plants. However, successful colonization is contingent upon the absence of the vertically transmitted symbiont or the impairment of its type VI secretion system. Unexpectedly for a vertically transmitted symbiosis, these findings suggest that microbe-microbe interactions, including antagonism, may play a crucial role in maintaining the specificity of an association. However, low transmission rates of synthetic symbionts provide evidence that transmission barriers or bottlenecks may still occur, further enforcing partner fidelity. Together, these results highlight the complexity of mechanisms underlying mutualistic associations, and provide insights into the evolution of bacterial leaf symbiosis.

Developmental transitions in the mosquito Aedes aegypti are central to vector competence and disease transmission, yet the underlying metabolic programs remain poorly defined. Here, we use untargeted metabolomics, gene expression analysis, and functional assays to delineate stage-specific metabolic fingerprints across the mosquito life cycle, from egg and larva to pupa and adult. Our profiling of the larval diet reveals comprehensive provisioning of essential nutrients, including B vitamins critical for development. Metabolomic analyses uncover distinct, stage-specific signatures, with the larval stage exhibiting a pronounced enrichment of methionine cycle metabolites and maximal methylation capacity. Notably, while S-adenosylmethionine (SAM) and related metabolites peak in larvae, the transcription of the methionine cycle and histone methyltransferase genes is highest in adults. Functional disruption of the methionine cycle in mosquito cells reveals network-level robustness and regulatory crosstalk within the pathway. However, we also identify a specific vulnerability: silencing the gene adenosylhomocysteinase (ahcy) enhances dengue virus 1 replication and infectious particle production. Collectively, our findings identify the methionine cycle as a metabolic epigenetic hub that integrates nutrition, development, and viral susceptibility, and highlight the larval stage as a strategic target for novel mosquito-control strategies. ### Competing Interest Statement The authors have declared no competing interest.

Aedes aegypti mosquitoes transmit arboviruses that pose a growing global health threat. After a bloodmeal, mosquitoes experience complex physiological changes orchestrated by the midgut and fat body, beginning with digestion and culminating in egg production. Our study provides comprehensive midgut and fat-body cell atlases using single-cell RNA sequencing and metabolomics. Our analyses reveal highly diverse cell populations specialized in digestion, metabolism, immunity, and reproduction. The midgut primarily comprises enterocytes, enteroendocrine, and intestinal stem cells, while the fat body features trophocytes and oenocytes but also a substantial hemocyte population and a newly found fat-body-yolk cell population. Additionally, Phasi Charoen-like virus was detected in midgut cells 7 days post bloodmeal. These findings highlight the complexity of mosquito abdominal tissues and inform the development of refined vector-control strategies, focusing on specific cell populations and metabolic pathways essential for mosquito reproductive success.

The impact of abiotic challenges on plant physiology reshapes plant-pathogen interactions by modulating the plant immune responses. In wheat, the development of Fusarium Head Blight (FHB) is heavily influenced by environmental conditions, especially during the pre-anthesis stage, just before fungal infection occurs. The early stages of infection are thus likely conditioned by prior environmental changes with consequences on the disease outcome that require further characterization. In this study, we aimed to assess the impact of pre-anthesis water depletion followed by rapid rehydration at inoculation on the expression of FHB-related molecular determinants with emphasis on susceptibility factors and metabolism-related processes. Water depletion altered plant physiology and its effects remained detectable after three days after rehydration, leading to significantly reduced FHB symptoms. Dual-transcriptomics, combined with untargeted metabolomics, revealed two key findings including (i) extensive metabolic changes specific to prior water stress, and (ii) the strong conservation of previously identified candidate susceptibility genes regulation. Considering the combined stress effects, a unique response signature emerged, highlighting that immune responses are strongly interwoven with physiological adjustments. Our findings provide new insights into the trade-offs that plants make under multiple challenges and identify original wheat metabolic determinants that may improve FHB resistance even in suboptimal physiological conditions.

Methanolic leaf (LME) and stem bark (SBME) extracts from Ficus carica L., were analysed using UHPLC-HRMS-MS analysis. Fifty-two compounds were identified belonging to flavonoids, phenolic acids and alkaloids. Their hepatoprotective activity was tested in a CCl4 rat model. In the CCl4-treated groups, the investigated extracts improved body weight (LME: 245.7 and SBME: 236.5 g) and relative liver weight (LME: 3.9 and SBME: 3.6 mg/g). They also reduced levels of aspartate aminotransferase (LME: 102.6 and SBME: 205.3.0 (UI/L), alanine aminotransferase (LME: 170.3 and SBME: 260.6 UI/L), lactate dehydrogenase (LME: 281.6 and SBME: 445.3 UI/L) and MDA (LME: 1.4 and SBME: 2.1 U/mg protein), while enhancing superoxide dismutase (LME: 65.1 and 46.2 U/mg protein) and catalase (LME: 11.0 and SBME: 8.3 U/mg protein) activities. These findings, together with histological examination, imply that the studied methanolic extracts protect the liver against steatosis, fibrosis and inflammation by increasing the antioxidative defense.

Esca is one of the main grapevine trunk diseases affecting vineyards worldwide. Phaeoacremonium minimum and Phaeomoniella chlamydospora are thought to be two of the main causal agents of this disease. However, the molecular mechanisms underlying plant defense responses in the grapevine trunk against esca-associated pathogens are poorly understood. To provide a first glimpse at the trunk responses to P. minimum and P. chlamydospora, transcriptomic and metabolomic analyses were performed to compare and contrast host responses to these pathogens. Transcriptomic analysis revealed different gene expression reprogramming in the trunk in response to each fungus. The main significant differences were found among genes associated with secondary metabolism, signaling, and hormone signaling. An untargeted liquid chromatography-high resolution mass spectrometry metabolomic approach performed 3 weeks after inoculation was used, and dereplication mainly highlighted flavonoids and stilbenes as plant defense metabolites in the infected trunk. Some metabolites were overproduced with both fungi, but specific responses were also observed. Particularly, a lipophilic flavonoid cluster was emphasized after P. minimum inoculation. The assessment of fungal infection 6 weeks postinfection showed more copies of P. minimum than P. chlamydospora. This dissimilarity in the level of colonization could be linked to the metabolomic responses observed. Our results reveal both different gene expression reprogramming and metabolomic-specific signatures depending on the wood pathogen. Altogether, these observations suggest that grapevine trunks can differently perceive and respond to P. minimum and P. chlamydospora. Copyright (c) 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Mosquito saliva plays a determining role in flavivirus transmission. Here, we discover and elucidate how salivary lipids enhance transmission. Building upon our discovery of salivary extracellular vesicles (EV), we determined that lipids within mosquito EVs, and neither within human EVs nor virions, enhance infection for flaviviruses in primary cell types relevant for transmission. Mechanistically, mosquito EV-lipids specifically promote viral protein levels by reducing ER-associated degradation. Infection enhancement is caused by sphingomyelins within mosquito salivary EVs that elevate sphingomyelin concentration within host cells. Transmission assays showed that mosquito EV-lipids exacerbate disease severity. Our study reveals that EV-associated sphingomyelins within mosquito saliva enhance transmission for multiple flaviviruses by reconfiguring the host lipidome to promote viral protein levels and the resulting skin infection. Our findings open a new dimension centered on lipids in the interplay between hosts, mosquitoes and flaviviruses that determine transmission, unveiling lipids as a new pan-flavivirus target. Highlights ### Competing Interest Statement The authors have declared no competing interest.

Open mass spectral libraries (OMSL) are critical for metabolite annotation and machine learning, especially given the rising volume of untargeted metabolomic studies and the development of annotation pipelines. Despite their importance, the practical application of OMSLs is hampered by the lack of standardized file formats, metadata fields, and supporting ontology. Current libraries, often restricted to specific topics or matrices such as natural products, lipids, or the human metabolome, may limit the discovery potential of untargeted studies. FragHub addresses these challenges by integrating multiple OMSLs into a single comprehensive database, supporting various data formats and harmonizing metadata. It also proposes some generic filters for mass spectrum using a graphical user interface. Additionally, a workflow to generate in-house libraries compatible with FragHub is proposed. FragHub dynamically segregates libraries based on ionization modes and chromatography techniques, thereby enhancing data utility in metabolomic research. The FragHub Python code is publicly available under a MIT license, at the following repository: https://github.com/eMetaboHUB/FragHub. Generated data can be accessed at https://doi.org/10.5281/zenodo.11057687.

Hura crepitans (Euphorbiaceae), is widespread in the Amazon rainforest and on plantations in sub-Saharan Africa. This tree produces an irritating milky latex rich in secondary metabolites, notably daphnane-type diterpenes and cerebrosides. Previous studies have shown that huratoxin, the main daphnane in the latex, significantly and selectively inhibited the growth of colorectal cancer cells through a unique mechanism involving the activation of PKCζ. One major challenge in isolating active molecules from natural products is the accessibility of the resource. This study explores the phytochemical composition and cytotoxic activities of latexes collected in Peru, Benin, and Togo using UHPLC-MS and metabolomics tools to identify a renewable source of bioactive compounds. Significant inter- and intra-continental differences in chemical composition have been highlighted, with daphnanes being concentrated in the Peruvian samples. Extracts form latexes collected in Peru showed cytostatic activity on Caco-2 cells, correlated with the presence of daphnanes, while some African samples exhibited cytotoxic activity on Jurkat and Hela cancer cell lines, leading to the identification of potential other new bioactive compounds such as elasterol and cerebrosides. OBJECTIVE: To compare the composition of different Hura crepitans latex samples and determine their cytotoxic activity in order to identify new bioactive compounds CONCLUSIONS: Inter- and intra-continental variations in the phytochemical composition of latex were observed, leading to significant cytotoxic activities on different cell lines. Daphnanes were identified as responsible for the activity on Caco-2 cells, while elasterol and cerebrosides were putatively associated with the activity on Hela cells.

ABSTRACT The rhizosphere, which serves as the primary interface between plant roots and the soil, constitutes an ecological niche for a huge diversity of microbial communities. Currently, there is little knowledge on the nature and the function of the different metabolites released by rhizospheric microbes to facilitate colonization of this highly competitive environment. Here, we demonstrate how the production of galbonolides, a group of polyene macrolides that inhibit plant and fungal Inositol Phosphorylceramide Synthase (IPCS), empowers the rhizospheric Streptomyces strain AgN23, to thrive in the rhizosphere by triggering the plant’s defence mechanisms. Metabolomic analysis of AgN23-inoculated Arabidopsis roots revealed a strong induction in the production of an indole alkaloid, camalexin, which is a major phytoalexin in Arabidopsis . By using a plant mutant compromised in camalexin synthesis, we show that camalexin production is necessary for the successful colonization of the rhizosphere by AgN23. Conversely, hindering galbonolides biosynthesis in AgN23 knock-out mutant resulted in loss of inhibition of IPCS, a deficiency in plant defence activation, notably the production of camalexin, and a strongly reduced development of the mutant bacteria in the rhizosphere. Together, our results identified galbonolides as important metabolites mediating rhizosphere colonisation by Streptomyces . Abstract Figure Graphical Abstract Model summarizing the mode of action of galbonolides in stimulating plant defence to support AgN23 colonization of the rhizosphere. Galbonolides secretion by Streptomyces sp. AgN23 trigger Inositol Phosphoceramide Synthase (IPCS) inhibition in Arabidopsis root cells (orange arrow). The resulting raise in Ceramide precursors of the IPCS may result in the different defence responses associated to AgN23: Hypersensitive Responses (HR), Salicylic Acid (SA) signalling, nuclear Ca 2+ influx, defence gene expression and camalexin biosynthesis. This production of camalexin (blue arrow) exert a positive effect on AgN23 growth in the rhizosphere, presumably by restricting the growth of bacterial and fungal competitors sensitive to this phytoalexin. In addition, galbonolides secretion in the rhizosphere may also directly interfere with fungal competitors of AgN23.