Ricca's factors as mobile proteinaceous effectors of electrical signaling.

Leaf-feeding insects trigger high-amplitude, defense-inducing electrical signals called slow wave potentials (SWPs). These signals are thought to be triggered by the long-distance transport of low molecular mass elicitors termed Ricca's factors. We sought mediators of leaf-to-leaf electrical signaling in Arabidopsis thaliana and identified them as ß-THIOGLUCOSIDE GLUCOHYDROLASE 1 and 2 (TGG1 and TGG2). SWP propagation from insect feeding sites was strongly attenuated in tgg1 tgg2 mutants and wound-response cytosolic Ca2+ increases were reduced in these plants. Recombinant TGG1 fed into the xylem elicited wild-type-like membrane depolarization and Ca2+ transients. Moreover, TGGs catalyze the deglucosidation of glucosinolates. Metabolite profiling revealed rapid wound-induced breakdown of aliphatic glucosinolates in primary veins. Using in vivo chemical trapping, we found evidence for roles of short-lived aglycone intermediates generated by glucosinolate hydrolysis in SWP membrane depolarization. Our findings reveal a mechanism whereby organ-to-organ protein transport plays a major role in electrical signaling.

Hyphenation of microflow chromatography with electrospray ionization mass spectrometry for bioanalytical applications focusing on low molecular weight compounds: A tutorial review.

Benefits of miniaturized chromatography with various detection modes, such as increased sensitivity, chromatographic efficiency, and speed, were recognized nearly 50 years ago. Over the past two decades, this approach has experienced rapid growth, driven by the emergence of mass spectrometry applications serving -omics sciences and the need for analyzing minute volumes of precious samples with ever higher sensitivity. While nanoscale liquid chromatography (flow rates <1 µL/min) has gained widespread recognition in proteomics, the adoption of microscale setups (flow rates ranging from 1 to 100 µL/min) for low molecular weight compound applications, including metabolomics, has been surprisingly slow, despite the inherent advantages of the approach. Highly heterogeneous matrices and chemical structures accompanied by a relative lack of options for both selective sample preparation and user-friendly equipment are usually reported as major hindrances. To facilitate the wider implementation of microscale analyses, we present here a comprehensive tutorial encompassing important theoretical and practical considerations. We provide fundamental principles in micro-chromatography and guide the reader through the main elements of a microflow workflow, from LC pumps to ionization devices. Finally, based on both our literature overview and experience, illustrated by some in-house data, we highlight the critical importance of the ionization source design and its careful optimization to achieve significant sensitivity improvement.

Photoreceptor-induced sinapate synthesis contributes to photoprotection in Arabidopsis.

Plants must balance light capture for photosynthesis with protection from potentially harmful ultraviolet radiation (UV). Photoprotection is mediated by concerted action of photoreceptors, but the underlying molecular mechanisms are not fully understood. In this study, we provide evidence that UV RESISTANCE LOCUS 8 (UVR8) UV-B-, phytochrome red-, and cryptochrome blue-light photoreceptors converge on the induction of FERULIC ACID 5-HYDROXYLASE 1 (FAH1) that encodes a key enzyme in the phenylpropanoid biosynthesis pathway, leading to the accumulation of UV-absorbing sinapate esters in Arabidopsis (Arabidopsis thaliana). FAH1 induction depends on the bZIP transcription factors ELONGATED HYPOCOTYL 5 (HY5) and HY5-HOMOLOG (HYH) that function downstream of all three photoreceptors. Noticeably, mutants with hyperactive UVR8 signaling rescue fah1 UV sensitivity. Targeted metabolite profiling suggests that this phenotypic rescue is due to the accumulation of UV-absorbing metabolites derived from precursors of sinapate synthesis, namely coumaroyl-glucose and feruloyl-glucose. Our genetic dissection of the phenylpropanoid pathway combined with metabolomic and physiological analyses show that both sinapate esters and flavonoids contribute to photoprotection with sinapates playing a major role for UV screening. Our findings indicate that photoreceptor-mediated regulation of FAH1 and subsequent accumulation of sinapate "sunscreen" compounds is a key protective mechanism to mitigate damage, preserving photosynthetic performance, and ensuring plant survival under UV.

Eight key rules for successful data-dependent acquisition in mass spectrometry-based metabolomics.

In recent years, metabolomics has emerged as a pivotal approach for the holistic analysis of metabolites in biological systems. The rapid progress in analytical equipment, coupled to the rise of powerful data processing tools, now provides unprecedented opportunities to deepen our understanding of the relationships between biochemical processes and physiological or phenotypic conditions in living organisms. However, to obtain unbiased data coverage of hundreds or thousands of metabolites remains a challenging task. Among the panel of available analytical methods, targeted and untargeted mass spectrometry approaches are among the most commonly used. While targeted metabolomics usually relies on multiple-reaction monitoring acquisition, untargeted metabolomics use either data-independent acquisition (DIA) or data-dependent acquisition (DDA) methods. Unlike DIA, DDA offers the possibility to get real, selective MS/MS spectra and thus to improve metabolite assignment when performing untargeted metabolomics. Yet, DDA settings are more complex to establish than DIA settings, and as a result, DDA is more prone to errors in method development and application. Here, we present a tutorial which provides guidelines on how to optimize the technical parameters essential for proper DDA experiments in metabolomics applications. This tutorial is organized as a series of rules describing the impact of the different parameters on data acquisition and data quality. It is primarily intended to metabolomics users and mass spectrometrists that wish to acquire both theoretical background and practical tips for developing effective DDA methods.

Legacy effects of premature defoliation in response to an extreme drought event modulate phytochemical profiles with subtle consequences for leaf herbivory in European beech.

Extreme droughts can have long-lasting effects on forest community dynamics and species interactions. Yet, our understanding of how drought legacy modulates ecological relationships is just unfolding. We tested the hypothesis that leaf chemistry and herbivory show long-term responses to premature defoliation caused by an extreme drought event in European beech (Fagus sylvatica L.). For two consecutive years after the extreme European summer drought in 2018, we collected leaves from the upper and lower canopy of adjacently growing drought-stressed and unstressed trees. Leaf chemistry was analyzed and leaf damage by different herbivore-feeding guilds was quantified. We found that drought had lasting impacts on leaf nutrients and on specialized metabolomic profiles. However, drought did not affect the primary metabolome. Drought-related phytochemical changes affected damage of leaf-chewing herbivores whereas damage caused by other herbivore-feeding guilds was largely unaffected. Drought legacy effects on phytochemistry and herbivory were often weaker than between-year or between-canopy strata variability. Our findings suggest that a single extreme drought event bears the potential to long-lastingly affect tree-herbivore interactions. Drought legacy effects likely become more important in modulating tree-herbivore interactions since drought frequency and severity are projected to globally increase in the coming decades.

Facilitative and competitive interactions between mycorrhizal and nonmycorrhizal plants in an extremely phosphorus-impoverished environment: role of ectomycorrhizal fungi and native oomycete pathogens in shaping species coexistence.

Nonmycorrhizal cluster root-forming species enhance the phosphorus (P) acquisition of mycorrhizal neighbours in P-impoverished megadiverse systems. However, whether mycorrhizal plants facilitate the defence of nonmycorrhizal plants against soil-borne pathogens, in return and via their symbiosis, remains unknown. We characterised growth and defence-related compounds in Banksia menziesii (nonmycorrhizal) and Eucalyptus todtiana (ectomycorrhizal, ECM) seedlings grown either in monoculture or mixture in a multifactorial glasshouse experiment involving ECM fungi and native oomycete pathogens. Roots of B. menziesii had higher levels of phytohormones (salicylic and jasmonic acids, jasmonoyl-isoleucine and 12-oxo-phytodienoic acid) than E. todtiana which further activated a salicylic acid-mediated defence response in roots of B. menziesii, but only in the presence of ECM fungi. We also found that B. menziesii induced a shift in the defence strategy of E. todtiana, from defence-related secondary metabolites (phenolic and flavonoid) towards induced phytohormone response pathways. We conclude that ECM fungi play a vital role in the interactions between mycorrhizal and nonmycorrhizal plants in a severely P-impoverished environment, by introducing a competitive component within the facilitation interaction between the two plant species with contrasting nutrient-acquisition strategies. This study sheds light on the interplay between beneficial and detrimental soil microbes that shape plant-plant interaction in severely nutrient-impoverished ecosystems.

Far-red light increases maize volatile emissions in response to volatile cues from neighbouring plants.

Plants perceive the presence and defence status of their neighbours through light and volatile cues, but how plants integrate both stimuli is poorly understood. We investigated if and how low Red to Far red light (R:FR) ratios, indicative of shading or canopy closure, affect maize (Zea mays) responses to herbivore-induced plant volatiles (HIPVs), including the green leaf volatile (Z)-3-hexenyl acetate. We modulated light signalling and perception by using FR supplementation and a phyB1phyB2 mutant, and we determined volatile release as a response readout. To gain mechanistic insights, we examined expression of volatile biosynthesis genes, hormone accumulation, and photosynthesis. Exposure to a full blend of HIPVs or (Z)-3-hexenyl acetate induced maize volatile release. Short-term FR supplementation increased this response. In contrast, prolonged FR supplementation or constitutive phytochrome B inactivation in phyB1phyB2 plants showed the opposite response. Short-term FR supplementation enhanced photosynthesis and stomatal conductance and (Z)-3-hexenyl acetate-induced JA-Ile levels. We conclude that a FR-enriched light environment can prompt maize plants to respond more strongly to HIPVs emitted by neighbours, which might be explained by changes in photosynthetic processes and phytochrome B signalling. Our findings reveal interactive responses to light and volatile cues with potentially important consequences for plant-plant and plant-herbivore interactions.

Spatial and evolutionary predictability of phytochemical diversity.

To cope with environmental challenges, plants produce a wide diversity of phytochemicals, which are also the source of numerous medicines. Despite decades of research in chemical ecology, we still lack an understanding of the organization of plant chemical diversity across species and ecosystems. To address this challenge, we hypothesized that molecular diversity is not only related to species diversity, but also constrained by trophic, climatic, and topographical factors. We screened the metabolome of 416 vascular plant species encompassing the entire alpine elevation range and four alpine bioclimatic regions in order to characterize their phytochemical diversity. We show that by coupling phylogenetic information, topographic, edaphic, and climatic variables, we predict phytochemical diversity, and its inherent composition, of plant communities throughout landscape. Spatial mapping of phytochemical diversity further revealed that plant assemblages found in low to midelevation habitats, with more alkaline soils, possessed greater phytochemical diversity, whereas alpine habitats possessed higher phytochemical endemism. Altogether, we present a general tool that can be used for predicting hotspots of phytochemical diversity in the landscape, independently of plant species taxonomic identity. Such an approach offers promising perspectives in both drug discovery programs and conservation efforts worldwide.

Consequences of squash (Cucurbita argyrosperma) domestication for plant defence and herbivore interactions.

MAIN CONCLUSION: Cucurbita argyrosperma domestication affected plant defence by downregulating the cucurbitacin synthesis-associated genes. However, tissue-specific suppression of defences made the cultivars less attractive to co-evolved herbivores Diabrotica balteata and Acalymma spp. Plant domestication reduces the levels of defensive compounds, increasing susceptibility to insects. In squash, the reduction of cucurbitacins has independently occurred several times during domestication. The mechanisms underlying these changes and their consequences for insect herbivores remain unknown. We investigated how Cucurbita argyrosperma domestication has affected plant chemical defence and the interactions with two herbivores, the generalist Diabrotica balteata and the specialist Acalymma spp. Cucurbitacin levels and associated genes in roots and cotyledons in three wild and four domesticated varieties were analysed. Domesticated varieties contained virtually no cucurbitacins in roots and very low amounts in cotyledons. Contrastingly, cucurbitacin synthesis-associated genes were highly expressed in the roots of wild populations. Larvae of both insects strongly preferred to feed on the roots of wild squash, negatively affecting the generalist's performance but not that of the specialist. Our findings illustrate that domestication results in tissue-specific suppression of chemical defence, making cultivars less attractive to co-evolved herbivores. In the case of squash, this may be driven by the unique role of cucurbitacins in stimulating feeding in chrysomelid beetles.

Novel insights into the contribution of plastoglobules and reactive oxygen species to chromoplast differentiation.

Plant tissues can be enriched in phytonutrients not only by stimulating their biosynthesis but also by providing appropriate sink structures for their sequestering and storage. In the case of carotenoids, they accumulate at high levels in chromoplasts naturally found in flowers and fruit. Chromoplasts can also be artificially differentiated from leaf chloroplasts by boosting carotenoid production with the bacterial protein crtB. Here we used electron and confocal microscopy together with subplastidial fractionation and transcript, protein and metabolite analyses to analyze the structural and biochemical changes occurring in crtB-induced artificial chromoplasts and their impact on the accumulation of health-related isoprenoids. We show that leaf chromoplasts develop plastoglobules (PG) harboring high levels of carotenoids (mainly phytoene and pro-vitamin A ß-carotene) but also other nutritionally relevant isoprenoids, such as tocopherols (vitamin E) and phylloquinone (vitamin K1). Further promoting PG proliferation by exposure to intense (high) light resulted in a higher accumulation of these health-related metabolites but also an acceleration of the chloroplast-to-chromoplast conversion. We further show that the production of reactive oxygen species (ROS) stimulates chromoplastogenesis. Our data suggest that carotenoid accumulation and ROS production are not just consequences but promoters of the chromoplast differentiation process.

Wound-response jasmonate dynamics in the primary vasculature.

The links between wound-response electrical signalling and the activation of jasmonate synthesis are unknown. We investigated damage-response remodelling of jasmonate precursor pools in the Arabidopsis thaliana leaf vasculature. Galactolipids and jasmonate precursors in primary veins from undamaged and wounded plants were analysed using MS-based metabolomics and NMR. In parallel, DAD1-LIKE LIPASEs (DALLs), which control the levels of jasmonate precursors in veins, were identified. A novel galactolipid containing the jasmonate precursor 12-oxo-phytodienoic acid (OPDA) was identified in veins: sn-2-O-(cis-12-oxo-phytodienoyl)-sn-3-O-(ß-galactopyranosyl) glyceride (sn-2-OPDA-MGMG). Lower levels of sn-1-OPDA-MGMG were also detected. Vascular OPDA-MGMGs, sn-2-18:3-MGMG and free OPDA pools were reduced rapidly in response to damage-activated electrical signals. Reduced function dall2 mutants failed to build resting vascular sn-2-OPDA-MGMG and OPDA pools and, upon wounding, dall2 produced less jasmonoyl-isoleucine (JA-Ile) than the wild-type. DALL3 acted to suppress excess JA-Ile production after wounding, whereas dall2 dall3 double mutants strongly reduce jasmonate signalling in leaves distal to wounds. LOX6 and DALL2 function to produce OPDA and the non-bilayer-forming lipid sn-2-OPDA-MGMG in the primary vasculature. Membrane depolarizations trigger rapid depletion of these molecules. We suggest that electrical signal-dependent lipid phase changes help to initiate vascular jasmonate synthesis in wounded leaves.

A glimmer of hope - ash genotypes with increased resistance to ash dieback pathogen show cross-resistance to emerald ash borer.

Plants rely on cross-resistance traits to defend against multiple, phylogenetically distinct enemies. These traits are often the result of long co-evolutionary histories. Biological invasions can force naïve plants to cope with novel, coincident pests, and pathogens. For example, European ash (Fraxinus excelsior) is substantially threatened by the emerald ash borer (EAB), Agrilus planipennis, a wood-boring beetle, and the ash dieback (ADB) pathogen, Hymenoscyphus fraxineus. Yet, plant cross-resistance traits against novel enemies are poorly explored and it is unknown whether naïve ash trees can defend against novel enemy complexes via cross-resistance mechanisms. To gain mechanistic insights, we quantified EAB performance on grafted replicates of ash genotypes varying in ADB resistance and characterized ash phloem chemistry with targeted and untargeted metabolomics. Emerald ash borer performed better on ADB-susceptible than on ADB-resistant genotypes. Moreover, changes in EAB performance aligned with differences in phloem chemical profiles between ADB-susceptible and ADB-resistant genotypes. We show that intraspecific variation in phloem chemistry in European ash can confer increased cross-resistance to invasive antagonists from different taxonomic kingdoms. Our study suggests that promotion of ADB-resistant ash genotypes may simultaneously help to control the ADB disease and reduce EAB-caused ash losses, which may be critical for the long-term stability of this keystone tree species.

Experimental Growth Conditions affect Direct and Indirect Defences in two Cotton Species.

Cotton has been used as a model plant to study direct and indirect plant defence against herbivorous insects. However, the plant growing conditions could have an important effect on the outcome of such plant defence studies. We examined how common experimental growth conditions influence constitutive and inducible defences in two species of cotton, Gossypium hirsutum and G. herbaceum. We induced plants by applying caterpillar regurgitant to mechanical wounds to compare the induction levels between plants of both species grown in greenhouse or phytotron conditions. For this we measured defence metabolites (gossypol and heliocides) and performance of Spodoptera frugiperda caterpillars on different leaves, the emission of plant volatiles, and their attractiveness to parasitic wasps. Induction increased the levels of defence metabolites, which in turn decreased the performance of S. frugiperda larvae. Constitutive and induced defence levels were the highest in plants grown in the phytotron (compared to greenhouse plants), G. hirsutum and young leaves. Defence induction was more pronounced in plants grown in the phytotron and in young leaves. Also, the differences between growing conditions were more evident for metabolites in the youngest leaves, indicating an interaction with plant ontogeny. The composition of emitted volatiles was different between plants from the two growth conditions, with greenhouse-grown plants showing more variation than phytotron-grown plants. Also, G. hirsutum released higher amounts of volatiles and attracted more parasitic wasps than G. herbaceum. Overall, these results highlight the importance of experimental abiotic factors in plant defence induction and ontogeny of defences. We therefore suggest careful consideration in selecting the appropriate experimental growing conditions for studies on plant defences.

A receptor-like protein mediates plant immune responses to herbivore-associated molecular patterns.

Herbivory is fundamental to the regulation of both global food webs and the extent of agricultural crop losses. Induced plant responses to herbivores promote resistance and often involve the perception of specific herbivore-associated molecular patterns (HAMPs); however, precisely defined receptors and elicitors associated with herbivore recognition remain elusive. Here, we show that a receptor confers signaling and defense outputs in response to a defined HAMP common in caterpillar oral secretions (OS). Staple food crops, including cowpea (Vigna unguiculata) and common bean (Phaseolus vulgaris), specifically respond to OS via recognition of proteolytic fragments of chloroplastic ATP synthase, termed inceptins. Using forward-genetic mapping of inceptin-induced plant responses, we identified a corresponding leucine-rich repeat receptor, termed INR, specific to select legume species and sufficient to confer inceptin-induced responses and enhanced defense against armyworms (Spodoptera exigua) in tobacco. Our results support the role of plant immune receptors in the perception of chewing herbivores and defense.

Genome-wide association mapping reveals genes underlying population-level metabolome diversity in a fungal crop pathogen.

BACKGROUND: Fungi produce a wide range of specialized metabolites (SMs) involved in biotic interactions. Pathways for the production of SMs are often encoded in clusters of tightly arranged genes identified as biosynthetic gene clusters. Such gene clusters can undergo horizontal gene transfers between species and rapid evolutionary change within species. The acquisition, rearrangement, and deletion of gene clusters can generate significant metabolome diversity. However, the genetic basis underlying variation in SM production remains poorly understood. RESULTS: Here, we analyzed the metabolite production of a large population of the fungal pathogen of wheat, Zymoseptoria tritici. The pathogen causes major yield losses and shows variation in gene clusters. We performed untargeted ultra-high performance liquid chromatography-high resolution mass spectrometry to profile the metabolite diversity among 102 isolates of the same species. We found substantial variation in the abundance of the detected metabolites among isolates. Integrating whole-genome sequencing data, we performed metabolite genome-wide association mapping to identify loci underlying variation in metabolite production (i.e., metabolite-GWAS). We found that significantly associated SNPs reside mostly in coding and gene regulatory regions. Associated genes encode mainly transport and catalytic activities. The metabolite-GWAS identified also a polymorphism in the 3'UTR region of a virulence gene related to metabolite production and showing expression variation. CONCLUSIONS: Taken together, our study provides a significant resource to unravel polymorphism underlying metabolome diversity within a species. Integrating metabolome screens should be feasible for a range of different plant pathogens and help prioritize molecular studies.

Nutritional stress exacerbates impact of a novel insecticide on solitary bees' behaviour, reproduction and survival.

Pesticide exposure and food stress are major threats to bees, but their potential synergistic impacts under field-realistic conditions remain poorly understood and are not considered in current pesticide risk assessments. We conducted a semi-field experiment to examine the single and interactive effects of the novel insecticide flupyradifurone (FPF) and nutritional stress on fitness proxies in the solitary bee Osmia bicornis. Individually marked bees were released into flight cages with monocultures of buckwheat, wild mustard or purple tansy, which were assigned to an insecticide treatment (FPF or control) in a crossed design. Nutritional stress, which was high in bees foraging on buckwheat, intermediate on wild mustard and low on purple tansy, modulated the impact of insecticide exposure. Within the first day after application of FPF, mortality of bees feeding on buckwheat was 29 times higher compared with control treatments, while mortality of FPF exposed and control bees was similar in the other two plant species. Moreover, we found negative synergistic impacts of FPF and nutritional stress on offspring production, flight activity, flight duration and flower visitation frequency. These results reveal that environmental policies and risk assessment schemes that ignore interactions among anthropogenic stressors will fail to adequately protect bees and the pollination services they provide.

Leafminer attack accelerates the development of soil-dwelling conspecific pupae via plant-mediated changes in belowground volatiles.

Herbivore population dynamics are strongly influenced by the interactions established through their shared host. Such plant-mediated interactions can occur between different herbivore species and different life developmental stages of the same herbivore. However, whether these interactions occur between leaf-feeding herbivores and their soil-dwelling pupae is unknown. We studied whether tomato (Solanum lycopersicum) leaf herbivory by the American serpentine leafminer Liriomyza trifolii affects the performance of conspecific pupae exposed to the soil headspace of the plant. To gain mechanistic insights, we performed insect bioassays with the jasmonate-deficient tomato mutant def-1 and its wild-type, along with phytohormones, gene expression and root volatiles analyses. Belowground volatiles accelerated leafminer metamorphosis when wild-type plants were attacked aboveground by conspecifics. The opposite pattern was observed for def-1 plants, in which aboveground herbivory slowed metamorphosis. Leafminer attack induced jasmonate and abscisic acid accumulation and modulated volatile production in tomato roots in a def-1-dependent manner. Our results demonstrate that aboveground herbivory triggers changes in root defence signalling and expression, which can directly or indirectly via changes in soil or microbial volatiles, alter pupal development time. This finding expands the repertoire of plant-herbivore interactions to herbivory-induced modulation of metamorphosis, with potential consequences for plant and herbivore community dynamics.

Molecular Dissection of Early Defense Signaling Underlying Volatile-Mediated Defense Regulation and Herbivore Resistance in Rice.

Herbivore-induced plant volatiles prime plant defenses and resistance, but how they are integrated into early defense signaling and whether a causal relationship exists between volatile defense priming and herbivore resistance is unclear. Here, we investigated the impact of indole, a common herbivore-induced plant volatile and modulator of many physiological processes in plants, bacteria, and animals, on early defense signaling and herbivore resistance in rice (Oryza sativa). Rice plants infested by fall armyworm (Spodoptera frugiperda) caterpillars release indole at a rate of up to 25 ng*h-1 Exposure to equal doses of exogenous indole enhances rice resistance to S. frugiperda Screening of early signaling components revealed that indole pre-exposure directly enhances the expression of the leucine-rich repeat-receptor-like kinase OsLRR-RLK1 Pre-exposure to indole followed by simulated herbivory increases (i.e. primes) the transcription, accumulation, and activation of the mitogen-activated protein kinase OsMPK3 and the expression of the downstream WRKY transcription factor gene OsWRKY70 as well as several jasmonate biosynthesis genes, resulting in higher jasmonic acid (JA) accumulation. Analysis of transgenic plants defective in early signaling showed that OsMPK3 is required and that OsMPK6 and OsWRKY70 contribute to indole-mediated defense priming of JA-dependent herbivore resistance. Therefore, herbivore-induced plant volatiles increase plant resistance to herbivores by positively regulating early defense signaling components.

Multiple neonicotinoids in children's cerebro-spinal fluid, plasma, and urine.

BACKGROUND: Neonicotinoids (NN) are selective neurotoxic pesticides that bind to insect but also mammal nicotinic acetycholine receptors (nAChRs). As the most widely used class of insecticides worldwide, they are ubiquitously found in the environment, wildlife, and foods, and thus of special concern for their impacts on the environment and human health. nAChRs are vital to proper brain organization during the prenatal period and play important roles in various motor, emotional, and cognitive functions. Little is known on children's contamination by NN. In a pilot study we tested the hypothesis that children's cerebro-spinal fluid (CSF) can be contaminated by NN. METHODS: NN were analysed in leftover CSF, blood, and urine samples from children treated for leukaemias and lymphomas and undergoing therapeutic lumbar punctions. We monitored all neonicotinoids approved on the global market and some of their most common metabolites by ultra-high performance liquid chromatography-tandem mass spectrometry. RESULTS: From August to December 2020, 14 children were consecutively included in the study. Median age was 8 years (range 3-18). All CSF and plasma samples were positive for at least one NN. Nine (64%) CSF samples and 13 (93%) plasma samples contained more than one NN. Thirteen (93%) CSF samples had N-desmethyl-acetamiprid (median concentration 0.0123, range 0.0024-0.1068 ng/mL), the major metabolite of acetamiprid. All but one urine samples were positive for ≥ one NN. A statistically significant linear relationship was found between plasma/urine and CSF N-desmethyl-acetamiprid concentrations. CONCLUSIONS: We have developed a reliable analytical method that revealed multiple NN and/or their metabolites in children's CSF, plasma, and urine. Our data suggest that contamination by multiple NN is not only an environmental hazard for non-target insects such as bees but also potentially for children.

Phosphate Suppression of Arbuscular Mycorrhizal Symbiosis Involves Gibberellic Acid Signaling.

Most land plants entertain a mutualistic symbiosis known as arbuscular mycorrhiza with fungi (Glomeromycota) that provide them with essential mineral nutrients, in particular phosphate (Pi), and protect them from biotic and abiotic stress. Arbuscular mycorrhizal (AM) symbiosis increases plant productivity and biodiversity and is therefore relevant for both natural plant communities and crop production. However, AM fungal populations suffer from intense farming practices in agricultural soils, in particular Pi fertilization. The dilemma between natural fertilization from AM symbiosis and chemical fertilization has raised major concern and emphasizes the need to better understand the mechanisms by which Pi suppresses AM symbiosis. Here, we test the hypothesis that Pi may interfere with AM symbiosis via the phytohormone gibberellic acid (GA) in the Solanaceous model systems Petunia hybrida and Nicotiana tabacum. Indeed, we find that GA is inhibitory to AM symbiosis and that Pi may cause GA levels to increase in mycorrhizal roots. Consistent with a role of endogenous GA as an inhibitor of AM development, GA-defective N. tabacum lines expressing a GA-metabolizing enzyme (GA methyltransferase-GAMT) are colonized more quickly by the AM fungus Rhizoglomus irregulare, and exogenous Pi is less effective in inhibiting AM colonization in these lines. Systematic gene expression analysis of GA-related genes reveals a complex picture, in which GA degradation by GA2 oxidase plays a prominent role. These findings reveal potential targets for crop breeding that could reduce Pi suppression of AM symbiosis, thereby reconciling the advantages of Pi fertilization with the diverse benefits of AM symbiosis.