B-GATA factors are required to repress high-light stress responses in Marchantia polymorpha and Arabidopsis thaliana.

GATAs are evolutionarily conserved zinc-finger transcription factors from eukaryotes. In plants, GATAs can be subdivided into four classes, A-D, based on their DNA-binding domain, and into further subclasses based on additional protein motifs. B-GATAs with a so-called leucine-leucine-methionine (LLM)-domain can already be found in algae. In angiosperms, the B-GATA family is expanded and can be subdivided in to LLM- or HAN-domain B-GATAs. Both, the LLM- and the HAN-domain are conserved domains of unknown biochemical function. Interestingly, the B-GATA family in the liverwort Marchantia polymorpha and the moss Physcomitrium patens is restricted to one and four family members, respectively. And, in contrast to vascular plants, the bryophyte B-GATAs contain a HAN- as well as an LLM-domain. Here, we characterise mutants of the single B-GATA from Marchantia polymorpha. We reveal that this mutant has defects in thallus growth and in gemma formation. Transcriptomic studies uncover that the B-GATA mutant displays a constitutive high-light (HL) stress response, a phenotype that we then also confirm in mutants of Arabidopsis thaliana LLM-domain B-GATAs, suggesting that the B-GATAs have a protective role towards HL stress.

Organic Amendments in a Long-term Field Trial-Consequences for the Bulk Soil Bacterial Community as Revealed by Network Analysis.

This study intended to elucidate the long-term effects of organic soil amendments on bacterial co-occurrence in bulk soil with and without addition of mineral fertiliser. Previous research mostly neglected the bacterial co-occurrence structure and focussed mainly on the parameters species diversity and abundance changes of species. Here we present a systematic comparison of two frequently used soil amendments, manure and straw, with regard to their impact on bacterial co-occurrence in a long-term field trial in Speyer, Germany. The approach involved 16S amplicon sequencing in combination with a bacterial network analysis, comparing the different fertiliser regimes. The results show an increase of bacterial diversity as well as an accumulation of bacteria of the order Bacillales in plots fertilised with manure compared to a control treatment. In the straw-amended plots neither an increase in diversity was found nor were indicative species detectable. Furthermore, network analysis revealed a clear impact of mineral fertiliser addition on bacterial co-occurrence structure. Most importantly, both organic amendments increased network complexity irrespective of mineral fertilisation regime. At the same time, the effects of manure and straw exhibited differences that might be explained by differences in their nutritional/chemical contents. It is concluded that bacterial interactions are a crucial parameter for the assessment of amendment effects regarding soil health and sustainability.

HCH phytoremediation potential of native plant species from a contaminated urban site in Turda, Romania.

Current physical or chemical methods used for remediation of soils contaminated with hexachlocyclohexane (HCH), leave behind significant levels of pollutants. Given the compound's volatility and persistence in the environment, sites contaminated with HCH remain a concern for the population living in nearby areas. By making use of both the recovery capacity and the pollutant uptake ability of spontaneously growing vegetation, our study aimed to identify native plant species able to cover and moreover take up the HCH left at a former lindane production unit in Turda, Romania. The results showed that dominant species across the study site like Lotus tenuis, Artemisia vulgaris or Tanacetum vulgare, were capable of taking up HCH in their tissues, according to different patterns that combined at the scale of the plant community. Regardless of the proximity of the HCH contamination hotspots, the development of the plant cover was characteristic for vegetation succession on disturbed soils of the Central European region. Finally, we conclude that plant species which grow spontaneously at the HCH contaminated site in Turda and are capable of taking up the pollutant, represent a self-sustainable and low maintenance phytomanagement approach that would allow for the reintegration of the site in the urban or industrial circuit and nevertheless would reduce the toxicity risk to the neighboring human inhabitants.

Small Molecules Inspired by the Natural Product Withanolides as Potent Inhibitors of Wnt Signaling.

Wnt signaling is a fundamental pathway that drives embryonic development and is essential for stem cell maintenance and tissue homeostasis. Dysregulation of Wnt signaling is linked to various diseases, and a constitutively active Wnt pathway drives tumorigenesis. Thus, disruption of the Wnt response is deemed a promising strategy for cancer drug discovery. However, only few clinical drug candidates that target Wnt signaling are available so far, and new small-molecule modulators of Wnt-related processes are in high demand. Here we describe the synthesis of small molecules inspired by withanolide natural products by using a pregnenolone-derived ß-lactone as the key intermediate that was transformed into a δ-lactone appended to the D-ring of the steroidal scaffold. This natural-product-inspired compound library contained potent inhibitors of Wnt signaling that act upstream of the destruction complex to stabilize Axin in a tankyrase-independent manner.

Metabolism of Ibuprofen by Phragmites australis: Uptake and Phytodegradation.

This study explores ibuprofen (IBP) uptake and transformation in the wetland plant species Phragmites australis and the underlying mechanisms. We grew P. australis in perlite under greenhouse conditions and treated plants with 60 µg/L of IBP. Roots and rhizomes (RR), stems and leaves (SL), and liquid samples were collected during 21 days of exposure. Results show that P. australis can take up, translocate, and degrade IBP. IBP was completely removed from the liquid medium after 21 days with a half-life of 2.1 days. IBP accumulated in RR and was partly translocated to SL. Meanwhile, four intermediates were detected in the plant tissues: hydroxy-IBP, 1,2-dihydroxy-IBP, carboxy-IBP and glucopyranosyloxy-hydroxy-IBP. Cytochrome P450 monooxygenase was involved in the production of the two hydroxy intermediates. We hypothesize that transformation of IBP was first catalyzed by P450, and then by glycosyltransferase, followed by further storage or metabolism in vacuoles or cell walls. No significant phytotoxicity was observed based on relative growth of plants and stress enzyme activities. In conclusion, we demonstrated for the first time that P. australis degrades IBP from water and is therefore a suitable species for application in constructed wetlands to clean wastewater effluents containing IBP and possibly also other micropollutants.

Synthesis of an Iridoid-Inspired Compound Collection and Discovery of Autophagy Inhibitors.

Iridoids comprise a large group of monoterpenoid natural products displaying a diverse array of biological activities ranging from neurotrophic to anti-inflammatory and anti-tumorigenic properties. Therefore, the development of concise synthesis routes to compound collections inspired by the structural features of these natural products is of particular relevance for chemical biology and medicinal chemistry. Herein we describe a samarium diiodide-mediated synthesis of a small, focused iridoid-inspired compound collection. Characterization of these iridoid analogues in biological assays revealed novel small-molecule inhibitors of autophagy.

Neuritogenic militarinone-inspired 4-hydroxypyridones target the stress pathway kinase MAP4K4.

Progressive loss and impaired restoration of neuronal activity are hallmarks of neurological diseases, and new small molecules with neurotrophic activity are in high demand. The militarinone alkaloids and structurally simplified analogues with 4-hydroxy-2-pyridone core structure induce pronounced neurite outgrowth, but their protein target has not been identified. Reported herein is the synthesis of a militarinone-inspired 4-hydroxy-2-pyridone collection, its investigation for enhancement of neurite outgrowth, and the discovery of the stress pathway kinase MAP4K4 as a target of the discovered neuritogenic pyridones. The most potent 4-hydroxy-2-pyridone is a selective ATP-competitive inhibitor of MAP4K4 but not of the other stress pathway related kinases, as proven by biochemical analysis and by a crystal structure of the inhibitor in complex with MAP4K4. The findings support the notion that MAP4K4 may be a new target for the treatment of neurodegenerative diseases.

N-acyl-homoserine lactone uptake and systemic transport in barley rest upon active parts of the plant.

Bacteria communicate with each other in a population density-dependent process known as quorum sensing. N-acyl-homoserine lactones (HSLs) are the autoinducers of Gram-negative bacteria and the best-studied quorum sensing signals so far. HSLs induce various responses in plants, including systemic resistance and root development. Here, we used different methods, including tritium labelling, sensor strain assays and monoclonal antibodies (mAbs), to analyse the uptake and translocation of C8- and C10- homoserine lactones into barley (Hordeum vulgare cv Barke). Both HSLs were already systemically transported into the shoot at 2 h after application. HSL uptake could be inhibited by orthovanadate, demonstrating that ABC transporters are involved in the uptake. Root transport occurs predominantly via the central cylinder, which was shown by transport inhibition via KCl application and autoradiography of root cross-sections. Furthermore, a newly established detection method with mAbs allowed the first detection of a systemic transport of long-chain HSLs in plants. The coupled use of different HSL detection methods demonstrated that the uptake and transport of HSLs into barley does not occur passively, but relies, at least partially, on active processes in the plant.

Limited effects of crop foliar Si fertilization on a marginal soil under a future climate scenario.

Growing crops on marginal lands is a promising solution to alleviate the increasing pressure on agricultural land in Europe. Such crops will however be at the same time exposed to increased drought and pathogen prevalence, on already challenging soil conditions. Some sustainable practices, such as Silicon (Si) foliar fertilization, have been proposed to alleviate these two stress factors, but have not been tested under controlled, future climate conditions. We hypothesized that Si foliar fertilization would be beneficial for crops under future climate, and would have cascading beneficial effects on ecosystem processes, as many of them are directly dependent on plant health. We tested this hypothesis by exposing spring barley growing on marginal soil macrocosms (three with, three without Si treatment) to 2070 climate projections in an ecotron facility. Using the high-capacity monitoring of the ecotron, we estimated C, water, and N budgets of every macrocosm. Additionally, we measured crop yield, the biomass of each plant organ, and characterized bacterial communities using metabarcoding. Despite being exposed to water stress conditions, plants did not produce more biomass with the foliar Si fertilization, whatever the organ considered. Evapotranspiration (ET) was unaffected, as well as water quality and bacterial communities. However, in the 10-day period following two of the three Si applications, we measured a significant increase in C sequestration, when climate conditions where significantly drier, while ET remained the same. We interpreted these results as a less significant effect of Si treatment than expected as compared with literature, which could be explained by the high CO2 levels under future climate, that reduces need for stomata opening, and therefore sensitivity to drought. We conclude that making marginal soils climate proof using foliar Si treatments may not be a sufficient strategy, at least in this type of nutrient-poor, dry, sandy soil.

Resilience of barley (Hordeum vulgare) plants upon exposure to tramadol: Implication for the root-associated bacterial community and the antioxidative plant defence system.

Insufficiently treated reclaimed water can act as a source of contamination by introducing recalcitrant contaminants (e.g., pharmaceutical compounds) to various water bodies and/or agricultural soils after irrigation. Tramadol (TRD) is one of these pharmaceuticals that can be detected in influents and effluents of wastewater treatment plants, at discharge points as well as in surface waters in Europe. While the uptake of TRD by plants through irrigation water has been shown, plant responses towards this compound are still unclear. Therefore, this study aims to evaluate the effects of TRD on selected plant enzymes as well as on the root bacterial community structure. A hydroponic experiment was conducted to test the effects of TRD (100 µg L-1 TRD) on barley plants, at two harvesting time points after treatment. Accumulation of TRD in root tissues over time was observed reaching concentrations of 111.74 and 138.39 µg g-1 in total root FW after 12 and 24 days of exposure, respectively. Furthermore, noticeable inductions in guaiacol peroxidase (5.47-fold), catalase (1.83-fold) and glutathione S-transferase (3.23- and 2.09-fold) were recorded in roots of TRD-treated plants compared to controls after 24 days. A significant alteration in the beta diversity of root-associated bacteria due to TRD treatment was observed. Three amplicon sequence variants assigned to Hydrogenophaga, U. Xanthobacteraceae and Pseudacidovorax were differentially abundant in TRD-treated compared to control plants at both harvesting time points. This study reveals the resilience of plants through the induction of the antioxidative system and changes in the root-associated bacterial community to cope with the TRD metabolization/detoxification process.

Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E-cadherin to disrupt intercellular adhesion.

Mammalian and prokaryotic high-temperature requirement A (HtrA) proteins are chaperones and serine proteases with important roles in protein quality control. Here, we describe an entirely new function of HtrA and identify it as a new secreted virulence factor from Helicobacter pylori, which cleaves the ectodomain of the cell-adhesion protein E-cadherin. E-cadherin shedding disrupts epithelial barrier functions allowing H. pylori designed to access the intercellular space. We then designed a small-molecule inhibitor that efficiently blocks HtrA activity, E-cadherin cleavage and intercellular entry of H. pylori.

Plant GATA Factors: Their Biology, Phylogeny, and Phylogenomics.

GATA factors are evolutionarily conserved transcription factors that are found in animals, fungi, and plants. Compared to that of animals, the size of the plant GATA family is increased. In angiosperms, four main GATA classes and seven structural subfamilies can be defined. In recent years, knowledge about the biological role and regulation of plant GATAs has substantially improved. Individual family members have been implicated in the regulation of photomorphogenic growth, chlorophyll biosynthesis, chloroplast development, photosynthesis, and stomata formation, as well as root, leaf, and flower development. In this review, we summarize the current knowledge of plant GATA factors. Using phylogenomic analysis, we trace the evolutionary origin of the GATA classes in the green lineage and examine their relationship to animal and fungal GATAs. Finally, we speculate about a possible conservation of GATA-regulated functions across the animal, fungal, and plant kingdoms.

Removal of tramadol from water using Typha angustifolia and Hordeum vulgare as biological models: Possible interaction with other pollutants in short-term uptake experiments.

Tramadol (TRD) is widely detected in aquatic ecosystems as a result of massive abuse and insufficient removal from wastewater facilities. As a result, TRD can contaminate groundwater sources and/or agricultural soils. While TRD toxicity has been reported from aquatic biota, data about TRD detection in plants are scarce. Moreover, information regarding plant capability for TRD removal is lacking. To understand the fate of this opioid, we have investigated the uptake, translocation and removal capacity of TRD by plants, addressing short-term and long-term uptake. The uptake rates of TRD, in excised barley and cattail roots, were 5.18 and 5.79 µg g-1 root fresh weight day-1, respectively. However, TRD uptake was strongly inhibited after co-exposing these roots either with the drug venlafaxine (similar molecular structure as TRD) or with quinidine (an inhibitor of cellular organic cation transporters). When barley seedlings were exposed to TRD in a hydroponic experiment a removal efficiency up to 90% (within 15 days) was obtained, with bioconcentration and translocation factors close to 9 and 1, respectively. The combination of results from both plants and the inhibition observed after treatment with quinidine revealed that organic cation transporters may be involved in the uptake of TRD by plants.

The changes in Lemna minor metabolomic profile: A response to diclofenac incubation.

Metabolomics is an emerging approach that investigates the changes in the metabolome profile. In the present study, Lemna minor -considered as an experimental aquatic plant model- was incubated with 10 and 100 µM diclofenac (DCF) for 96 h, respectively. Knowing that DCF is internationally often problematic in wastewater effluents and that it might affect particularly the metabolic profiles in aquatic plants, mainly the oxidoreductase, dehydrogenase, peroxidase, and glutathione reductase activities, here it was hypothesized (H) that in the common duckweed, DCF might increase the phenolic and flavonoids pathways, as an antioxidant response to this stress (H1). Also, it was expected DCF to alternate the physiological characteristics, especially the molecular interaction and biochemical properties, of Lemna (H2). Metabolic changes were investigated with target and untargeted screening analysis using RPLC-HILIC-ESI-TOF-MS. Twelve amino acids were identified in all treatments, together with three organic acids (p-coumaric, cinnamic, and sinapic acids). In untargeted screening, the important metabolites to discriminate between different treatments were assigned to Lemna such as organic acids, lignin, sugars, amino acids, dipeptides, flavonoids, biflavonoids, fatty acids, among others. In resume, Lemna responded to both DCF concentrations, showing different stress patterns. A similar metabolic response had already been identified in other studies in exposing Lemna to other anthropogenic stressors (like pesticides).

Fate and impact of wastewater-borne micropollutants in lettuce and the root-associated bacteria.

The reuse of water for agricultural practices becomes progressively more important due to increasing demands for a transition to a circular economy. Treated wastewater can be an alternative option of blue water used for the irrigation of crops but its risks need to be evaluated. This study assesses the uptake and metabolization of pharmaceuticals and personal care products (PPCPs) derived from treated wastewater into lettuce as well as the impact on root-associated bacteria under a realistic and worst-case scenario. Lettuce was grown in a controlled greenhouse and irrigated with water or treated wastewater spiked with and without a mixture of fourteen different PPCPs at 10 µg/L or 100 µg/L. After harvesting the plants, the same soil was reused for a consecutive cultivation campaign to test for the accumulation of PPCPs. Twelve out of fourteen spiked PPCPs were detected in lettuce roots, and thirteen in leaves. In roots, highest concentrations were measured for sucralose, sulfamethoxazole and citalopram, while sucralose, acesulfame and carbamazepine were the highest in leaves. Higher PPCP concentrations were found in lettuce roots irrigated with spiked treated wastewater than in those irrigated with spiked water. The absolute bacterial abundance remained stable over both cultivation campaigns and was not affected by any of the treatments (type of irrigation water (water vs. wastewater) nor concentration of PPCPs). However, the irrigation of lettuce with treated wastewater had a significant effect on the microbial α-diversity indices at the end of the second cultivation campaign, and modified the structure and community composition of root-associated bacteria at the end of both campaigns. Five and fourteen bacterial families were shown to be responsible for the observed changes at the end of the first and second cultivation campaign, respectively. Relative abundance of Haliangium and the clade Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium was significantly affected in response to PCPPs exposure. Caulobacter, Cellvibrio, Hydrogenophaga and Rhizobacter were significantly affected in microcosms irrigated with wastewater.

Relaunch cropping on marginal soils by incorporating amendments and beneficial trace elements in an interdisciplinary approach.

In the EU and world-wide, agriculture is in transition. Whilst we just converted conventional farming imprinted by the post-war food demand and heavy agrochemical usage into integrated and sustainable farming with optimized production, we now have to focus on even smarter agricultural management. Enhanced nutrient efficiency and resistance to pests/pathogens combined with a greener footprint will be crucial for future sustainable farming and its wider environment. Future land use must embrace efficient production and utilization of biomass for improved economic, environmental, and social outcomes, as subsumed under the EU Green Deal, including also sites that have so far been considered as marginal and excluded from production. Another frontier is to supply high-quality food and feed to increase the nutrient density of staple crops. In diets of over two-thirds of the world's population, more than one micronutrient (Fe, Zn, I or Se) is lacking. To improve nutritious values of crops, it will be necessary to combine integrated, systems-based approaches of land management with sustainable redevelopment of agriculture, including central ecosystem services, on so far neglected sites: neglected grassland, set aside land, and marginal lands, paying attention to their connectivity with natural areas. Here we need new integrative approaches which allow the application of different instruments to provide us not only with biomass of sufficient quality and quantity in a site specific manner, but also to improve soil ecological services, e.g. soil C sequestration, water quality, habitat and soil resistance to erosion, while keeping fertilization as low as possible. Such instruments may include the application of different forms of high carbon amendments, the application of macro- and microelements to improve crop performance and quality as well as a targeted manipulation of the soil microbiome. Under certain caveats, the potential of such sites can be unlocked by innovative production systems, ready for the sustainable production of crops enriched in micronutrients and providing services within a circular economy.

Exudates from Miscanthus x giganteus change the response of a root-associated Pseudomonas putida strain towards heavy metals.

The composition of root exudates is modulated by several environmental factors, and it remains unclear how that affects beneficial rhizosphere or inoculated microorganisms under heavy metal (HM) contamination. Therefore, we evaluated the transcriptional response of Pseudomonas putida E36 (a Miscanthus x giganteus isolate with plant growth promotion-related properties) to Cd, Pb and Zn in an in vitro study implementing root exudates from M. x giganteus. To collect root exudates and analyse their composition plants were grown in a pot experiment under HM and control conditions. Our results indicated higher exudation rate for plants challenged with HM. Further, out of 29 organic acids identified and quantified in the root exudates, 8 of them were significantly influenced by HM (e.g., salicylic and terephthalic acid). The transcriptional response of P. putida E36 was significantly affected by the HM addition to the growth medium, increasing the expression of several efflux pumps and stress response-related functional units. The additional supplementation of the growth medium with root exudates from HM-challenged plants resulted in a downregulation of 29% of the functional units upregulated in P. putida E36 as a result of HM addition to the growth medium. Surprisingly, root exudates + HM downregulated the expression of P. putida E36 functional units related to plant colonization (e.g., chemotaxis, motility, biofilm formation) but upregulated its antibiotic and biocide resistance compared to the control treatment without HM. Our findings suggest that HM-induced changes in root exudation pattern may attract beneficial bacteria that are in turn awarded with organic nutrients, helping them cope with HM stress. However, it might affect the ability of these bacteria to colonize plants growing in HM polluted areas. Those findings may offer an insight for future in vivo studies contributing to improvements in phytoremediation measures.

Effect of the pharmaceuticals diclofenac and lamotrigine on stress responses and stress gene expression in lettuce (Lactuca sativa) at environmentally relevant concentrations.

Vegetable crops irrigated with treated wastewater can take up the environmentally persistent pharmaceuticals diclofenac and lamotrigine. This study aimed at quantifying the uptake and translocation of the two pharmaceuticals in lettuce (Lactuca sativa) as well as on the elucidation of the molecular and physiological changes triggered by them. Therefore, plants were cultivated in a phytochamber in hydroponic systems under controlled conditions and treated independently with diclofenac (20 µg L-1) and lamotrigine (60 µg L-1) for 48 h. A low translocation of lamotrigine but not of diclofenac or its metabolite 4'-hydroxydiclofenac to leaves was observed, which corresponded with the expression of stress related genes only in roots of diclofenac treated plants. We observed an oxidative burst in roots and leaves occurring around the same time point when lamotrigine was detected in leaves. This could be responsible for the significantly changed gene expression pattern in both tissues. Our results showed for the first time that pharmaceuticals like lamotrigine or diclofenac might act as signals or zeitgebers, affecting the circadian expression of stress related genes in lettuce possibly causing a repressed physiological status of the plant.

Post-reclamation microbial diversity and functions in hexachlorocyclohexane (HCH) contaminated soil in relation to spontaneous HCH tolerant vegetation.

The toxicity, volatility and persistence of the obsolete organochlorine pesticide hexachlorocyclohexane (HCH), makes reclamation of contaminated areas a priority for the health and welfare of neighboring human communities. Microbial diversity and functions and their relation to spontaneous vegetation in post-excavation situations, are essential indicators to consider in bioaugmentation or microbe-assisted phytoremediation strategies at field scale. Our study aimed to evaluate the effects of long-term HCH contamination on soil and plant-associated microbial communities, and whether contaminated soil has the potential to act as a bacterial inoculum in post-excavation bioremediation strategies. To scrutinize the role of vegetation, the potential nitrogen fixation of free-living and symbiotic diazotrophs of the legume Lotus tenuis was assessed as a measure of nutrient cycling functions in soil under HCH contamination. Potential nitrogen fixation was generally not affected by HCH, with the exception of lower nifH gene counts in excavated contaminated rhizospheres, most probably a short-term HCH effect on early bacterial succession in this compartment. HCH shaped microbial communities in long-term contaminated bulk soil, where we identified possible HCH tolerants such as Sphingomonas and Altererythrobacter. In L. tenuis rhizosphere, microbial community composition was additionally influenced by plant growth stage. Sphingobium and Massilia were the bacterial genera characteristic for HCH contaminated rhizospheres. Long-term HCH contamination negatively affected L. tenuis growth and development. However, root-associated bacterial community composition was driven solely by plant age, with negligible HCH effect. Results showed that L. tenuis acquired possible HCH tolerant bacteria such as the Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium clade, Sphingomonas, Massilia or Pantoea which could simultaneously offer plant growth promoting (PGP) benefits for the host. Finally, we identified an inoculum with possibly HCH tolerant, PGP bacteria transferred from the contaminated bulk soil to L. tenuis roots through the rhizosphere compartment, consisting of Mesorhizobium loti, Neorhizobium galegae, Novosphingobium lindaniclasticum, Pantoea agglomerans and Lysobacter bugurensis.

Untargeted Analysis of Lemna minor Metabolites: Workflow and Prioritization Strategy Comparing Highly Confident Features between Different Mass Spectrometers.

Metabolomics approaches provide a vast array of analytical datasets, which require a comprehensive analytical, statistical, and biochemical workflow to reveal changes in metabolic profiles. The biological interpretation of mass spectrometric metabolomics results is still obstructed by the reliable identification of the metabolites as well as annotation and/or classification. In this work, the whole Lemna minor (common duckweed) was extracted using various solvents and analyzed utilizing polarity-extended liquid chromatography (reversed-phase liquid chromatography (RPLC)-hydrophilic interaction liquid chromatography (HILIC)) connected to two time-of-flight (TOF) mass spectrometer types, individually. This study (introduces and) discusses three relevant topics for the untargeted workflow: (1) A comparison study of metabolome samples was performed with an untargeted data handling workflow in two different labs with two different mass spectrometers using the same plant material type. (2) A statistical procedure was observed prioritizing significant detected features (dependent and independent of the mass spectrometer using the predictive methodology Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA). (3) Relevant features were transferred to a prioritization tool (the FOR-IDENT platform (FI)) and were compared with the implemented compound database PLANT-IDENT (PI). This compound database is filled with relevant compounds of the Lemnaceae, Poaceae, Brassicaceae, and Nymphaceae families according to analytical criteria such as retention time (polarity and LogD (pH 7)) and accurate mass (empirical formula). Thus, an untargeted analysis was performed using the new tool as a prioritization and identification source for a hidden-target screening strategy. Consequently, forty-two compounds (amino acids, vitamins, flavonoids) could be recognized and subsequently validated in Lemna metabolic profile using reference standards. The class of flavonoids includes free aglycons and their glycosides. Further, according to our knowledge, the validated flavonoids robinetin and norwogonin were for the first time identified in the Lemna minor extracts.