N-containing Metabolites Research Articles

The dosage- and size-dependent effects of micro- and nanoplastics in lettuce roots and leaves at the growth, photosynthetic, and metabolomics levels

The occurrence of microplastics (MPs) and nanoplastics (NPs) in soils potentially induce morphological, physiological, and biochemical alterations in plants. The present study investigated the effects of MPs/NPs on lettuce (Lactuca sativa L. var. capitata) plants by focusing on (i) four different particle sizes of polyethylene micro- and nanoplastics, at (ii) four concentrations. Photosynthetic activity, morphological changes in plants, and metabolomic shifts in roots and leaves were investigated.Our findings revealed that particle size plays a pivotal role in influencing various growth traits of lettuce (biomass, color segmentation, greening index, leaf area, and photosynthetic activity), physiological parameters (including maximum quantum yield - Fv/Fmmax, or quantum yield in the steady-state Fv/FmLss, NPQLss, RfdLss, FtLss, FqLss), and metabolomic signatures. Smaller plastic sizes demonstrated a dose-dependent impact on aboveground plant structures, resulting in an overall elicitation of biosynthetic processes. Conversely, larger plastic size had a major impact on root metabolomics, leading to a negative modulation of biosynthetic processes. Specifically, the biosynthesis of secondary metabolites, phytohormone crosstalk, and the metabolism of lipids and fatty acids were among the most affected processes. In addition, nitrogen-containing compounds accumulated following plastic treatments.Our results highlighted a tight correlation between the qPCR analysis of genes associated with the soil nitrogen cycle (such as NifH, NirK, and NosZ), available nitrogen pools in soil (including NO3− and NH4), N-containing metabolites and morpho-physiological parameters of lettuce plants subjected to MPs/NPs. These findings underscore the intricate relationship between specific plastic contaminations, nitrogen dynamics, and plant performance.

Nitrogen deficiency stimulates cannabinoid biosynthesis in medical cannabis plants by inducing a metabolic shift towards production of low-N metabolites

The primary and secondary metabolism of plants is closely connected to the resources supplied and obtained by the plant, including their mineral nutrition. We recently reported that nitrogen (N) deficiency enhances the production of terpenoids and cannabinoids, the unique biologically-active secondary metabolites in medical cannabis plants. Knowledge-gaps concerning effects of N supply on primary metabolism in cannabis hinder understanding of the interrelations between N inputs and biosynthesis of the therapeutic secondary metabolites. The present study therefore evaluated the hypothesis that a decrease in the plant’s C (carbon): N ratio by N-limitation, induces a shift in the plant metabolism towards lower production of N-containing metabolites and higher production of metabolites that do not contain N. Effects of N input levels (30, 80, 160, 240, and 320 mg L−1) on total C and N concentrations and C: N ratio in leaves and inflorescences of medical cannabis plants were studied; and gas chromatography-mass spectrometry (GC-MS) analysis of primary metabolites added to the analyses of secondary metabolites. Elevation of N supply resulted in an increase in total N and N-containing compounds (chlorophylls and most amino acids), and decreased total C and compounds that do not contain N, such as sugars (fructose, glucose, and xylose), and phosphates (phosphate and glucose-6-phosphate) in both inflorescences and leaves. In the inflorescences, the elevation of N input also decreased total cannabinoids, phenols, and flavonoids, that do not contain N. Integrating the metabolic datasets revealed positive correlations between C sources (fructose and glucose) and most of the cannabinoids and terpenoids; the latter were negatively correlated with N-compounds (most amino acids). Taken together, these results suggest that elevated N supply induce a metabolic shift in the inflorescences towards increased production of N-compounds via deflecting the C sources from the biologically active compounds. In addition, the cannabis leaf was found to be more sensitive than the inflorescence to N supply, presenting greater changes in primary metabolism and more coordinated metabolic associations. These findings highlight the importance of adequate and precise N nutrition for standardization of the therapeutic-metabolite profile and for preventing undesirable metabolic repartitioning in medical cannabis plants.

Comparison of Balanites aegyptiaca parts: metabolome providing insights into plant health benefits and valorization purposes as analyzed using multiplex GC-MS, LC-MS, NMR-based metabolomics, and molecular networking.

Balanites aegyptiaca (L.) Delile (Zygophyllaceae), also known as the desert date, is an edible fruit-producing tree popular for its nutritional and several health benefits. In this study, multi-targeted comparative metabolic profiling and fingerprinting approaches were conducted for the assessment of the nutrient primary and secondary metabolite heterogeneity in different parts, such as leaves, stems, seeds, unripe, and ripe fruits of B. aegyptiaca using nuclear magnetic resonance (NMR), ultra-performance liquid chromatography (UPLC-MS), and gas chromatography mass-spectrometry (GC-MS) based metabolomics coupled to multivariate analyses and in relation to its cytotoxic activities. NMR-based metabolomic study identified and quantified 15 major primary and secondary metabolites belonging to alkaloids, saponins, flavonoids, sugars, and amino and fatty acids. Principal component analysis (PCA) of the NMR dataset revealed α-glucose, sucrose, and isorhamnetin as markers for fruit and stem and unsaturated fatty acids for predominated seeds. Orthogonal projections to latent structure discriminant analysis (OPLS-DA) revealed trigonelline as a major distinctive metabolite in the immature fruit and isorhamnetin as a major distinct marker in the mature fruit. UPLC-MS/MS analysis using feature-based molecular networks revealed diverse chemical classes viz. steroidal saponins, N-containing metabolites, phenolics, fatty acids, and lipids as the constitutive metabolome in Balanites. Gas chromatography-mass spectroscopy (GC-MS) profiling of primary metabolites led to the detection of 135 peaks belonging to sugars, fatty acids/esters, amino acids, nitrogenous, and organic acids. Monosaccharides were detected at much higher levels in ripe fruit and disaccharides in predominate unripe fruits, whereas B. aegyptiaca vegetative parts (leaves and stem) were rich in amino acids and fatty acids. The antidiabetic compounds, viz, nicotinic acid, and trigonelline, were detected in all parts especially unripe fruit in addition to the sugar alcohol d-pinitol for the first time providing novel evidence for B. aegyptiaca use in diabetes. In vitro cytotoxic activity revealed the potential efficacy of immature fruit and seeds as cytotoxic agents against human prostate cancer (PC3) and human colorectal cancer (HCT-116) cell lines. Collectively, such detailed profiling of parts provides novel evidence for B. aegyptiaca medicinal uses.

Integrated Metabolomics and Morpho-Biochemical Analyses Reveal a Better Performance of Azospirillum brasilense over Plant-Derived Biostimulants in Counteracting Salt Stress in Tomato.

Increased soil salinity is one of the main concerns in agriculture and food production, and it negatively affects plant growth and crop productivity. In order to mitigate the adverse effects of salinity stress, plant biostimulants (PBs) have been indicated as a promising approach. Indeed, these products have a beneficial effect on plants by acting on primary and secondary metabolism and by inducing the accumulation of protective molecules against oxidative stress. In this context, the present work is aimed at comparatively investigating the effects of microbial (i.e., Azospirillum brasilense) and plant-derived biostimulants in alleviating salt stress in tomato plants by adopting a multidisciplinary approach. To do so, the morphological and biochemical effects were assessed by analyzing the biomass accumulation and root characteristics, the activity of antioxidant enzymes and osmotic stress protection. Furthermore, modifications in the metabolomic profiles of both leaves and root exudates were also investigated by ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS). According to the results, biomass accumulation decreased under high salinity. However, the treatment with A. brasilense considerably improved root architecture and increased root biomass by 156% and 118% in non-saline and saline conditions, respectively. The antioxidant enzymes and proline production were enhanced in salinity stress at different levels according to the biostimulant applied. Moreover, the metabolomic analyses pointed out a wide set of processes being affected by salinity and biostimulant interactions. Crucial compounds belonging to secondary metabolism (phenylpropanoids, alkaloids and other N-containing metabolites, and membrane lipids) and phytohormones (brassinosteroids, cytokinins and methylsalicylate) showed the most pronounced modulation. Overall, our results suggest a better performance of A. brasilense in alleviating high salinity than the vegetal-derived protein hydrolysates herein evaluated.

Identification of Oxindoleacetic Acid Conjugates in Quinoa (Chenopodium quinoa Willd.) Seeds by High-Resolution UHPLC-MS/MS.

Quinoa (Chenopodium quinoa Willd.) has a high nutritional value and it contains a high number and high amounts of specialized metabolites. These metabolites include, for example, phenolic acids, flavonoids, terpenoids and steroids. In addition, it is known to contain N-containing metabolites, such as betalains. Here, we report the presence and identification of 14 new oxindoleacetate conjugates in quinoa by high-resolution ultrahigh-performance liquid chromatography quadrupole-time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) and ultrahigh-resolution UHPLC-QOrbitrap-MS/MS. The oxindoleacetate conjugates were extracted from dried and ground quinoa seeds using either methanol/water or acetone/water (4:1, v/v) and were further concentrated into aqueous phase and analyzed by UHPLC with reverse-phase chromatography using acetonitrile and 0.1% aqueous formic acid as eluents. High-resolution hybrid LC-MS/MS techniques, including full scan MS with in-source collision, induced dissociation, and data dependent-MS2(TopN) with stepped normalized collision energies using N2 as collision gas and data-independent acquisition (MSE) using ramped collision energies and argon as collision gas enabled their analysis directly from the crude quinoa seed extract. The oxindoleacetate conjugates were found to be present in both conventional and organic farmed seeds. According to our best knowledge, this is the first time hydroxy-oxindoles have been reported in quinoa.

Growth and biochemical profiling of marine microalgae Chlorella salina with response to nitrogen starvation

ABSTRACT The present study aimed at investigating nitrogen stress-induced biochemical changes in the marine microalgae Chlorella salina. The algae were grown in N free medium (N − ) in order to study the proximate composition via the determination of protein, lipid and carbohydrate. The chemical constituents of this algae were determined using GC-MS and mineral content was analysed using Inductive Coupled Plasma-Mass Spectrometry (ICP-MS). From the results, the proximate analysis showed increased lipid (34.77 ± 0.6%), followed by carbohydrate (26.21 ± 0.2%) and protein (5.4% dry weight). The chemical constituents of C. salina were successively extracted with diethyl ether, hexane, chloroform, acetone and methanol. The extract percentage varied greatly between various solvents, with the highest one (4.68%) recorded for methanol. A total of 35 compounds were identified from the extracts and the identified chemical groups included ketones, terpenes, fatty acids (FA), fatty alcohols and esters derived fatty acids and/or dicarboxylic acid and hydrocarbons. The methanol extract consisted of FA (35%), amino acids and N-containing metabolites (7%), fatty alcohols (8%), organic acids (3%) and hydrocarbons (80%). The algae exhibited high amounts of Mg (54.079 mg/50 g) followed by C (17.341 mg/50 g), S (5.702 mg/50 g) and Na (3.275 mg/50 g). These results demonstrated a strong correlation between the nitrogen content and lipid accumulation, hence showing that Chlorella salina could serve as a potential candidate for biodiesel production.

LC-MS-Based Metabolomics Discriminates Premium from Standard Chilean cv. Cabernet Sauvignon Wines from Different Valleys.

Cabernet Sauvignon grapes in Chile, mainly grown between the 30° S and 36° S, account for more than 30% of Chilean wine production, and yield wines with different characteristics which influence their quality. The aim of this study was to apply a liquid chromatography – mass spectrometry (LC–MS)-based metabolomic protocol to investigate the quality differentiation in a sample set of monovarietal wines from eight valleys covering 679 km of the north-south extension. All samples were produced using a standardized red winemaking process and classified according to a company categorization in two major groups: premium and standard, and each group in two subcategories. The results pointed out that N-containing metabolites (mainly small peptides) are promising biomarkers for quality differentiation. Moreover, the premium wines were characterized by higher amounts of anthocyanins and other glycosylated and acetylated flavonoids, as well as phenolic acids; standard quality wines, on the other hand, presented stilbenoids and sulfonated catabolites of tryptophan and flavanols.

Light spectral composition affects metabolic response and flowering in non-vernalized Ranunculus asiaticus L.

We investigated the influence of photoperiodic light spectrum, inducing different phytochrome photoequilibria (PPE) at plant level, on photosynthesis, metabolic profiling, plant growth and flowering of Ranunculus asiaticus L. hybrids, MBO and MDR with different flowering earliness, grown in glasshouse from rehydrated dry tuberous roots. Plants were exposed to three photoperiodic treatments (day extension to 14 h), compared to natural day length (NL): white fluorescent light (PPE 0.84), and LEDs Red:Far Red light at 3:1 ratio (PPE 0.84) and 1:3 ratio (PPE 0.63). We discuss the results also compared to data on plants from rehydrated and vernalized roots previously reported in Modarelli et al., 2000a.Leaf gas exchanges and quantum yield of PSII electron transport were higher in MDR than in MBO, whereas non-photochemical quenching showed the opposite behaviour. In MDR, R:FR 3:1 light was the most effective in promoting stomatal conductance, while it reduced photochemistry and increased heat dissipation compared to other treatments. Under NL, leaf area was greater in MBO while flowering earliness and flower stems were similar in the hybrids. Photoperiodic treatments did not influence the plant growth while anticipated flowering in both the hybrids.In both the hybrids, lighting did not change the content of chlorophylls, carotenoids, glucose and sucrose. The greater number and expansion of leaves in MBO, except under R:FR 3:1, was accompanied by a lower photosynthetic capacity per leaf area. Conversely, in MDR, with lower leaf number, area and DW, an interesting increase of N-containing metabolites (i.e. chlorophylls and amino acids) occurred, thus exerting a positive effect on photosynthetic rate.

Metabolite Profiling and Network Analysis Reveal Coordinated Changes in Low-N Tolerant and Low-N Sensitive Maize Genotypes under Nitrogen Deficiency and Restoration Conditions.

Nitrogen (N), applied in the form of a nitrogenous fertilizer, is one of the main inputs for agricultural production. Food production is closely associated with the application of N. However, the application of nitrogenous fertilizers to agricultural fields is associated with heavy production of nitrous oxide because agricultural crops can only utilize 30–40% of applied N, leaving behind unused 60–70% N in the environment. The global warming effect of this greenhouse gas is approximately 300 times more than of carbon dioxide. Under the present scenario of climate change, it is critical to maintain the natural balance between food production and environmental sustainability by targeting traits responsible for improving nitrogen-use-efficiency (NUE). Understanding of the molecular mechanisms behind the metabolic alterations due to nitrogen status needs to be addressed. Additionally, mineral nutrient deficiencies and their associated metabolic networks have not yet been studied well. Given this, the alterations in core metabolic pathways of low-N tolerant (LNT) and low-N sensitive (LNS) genotypes of maize under N-deficiency and their efficiency of recovering the changes upon resupplying N were investigated by us, using the GC–MS and LC–MS based metabolomic approach. Significant genotype-specific changes were noted in response to low-N. The N limitation affected the whole plant metabolism, most significantly the precursors of primary metabolic pathways. These precursors may act as important targets for improving the NUE. Limited availability of N reduced the levels of N-containing metabolites, organic acids and amino acids, but soluble sugars increased. Major variations were encountered in LNS, as compared to LNT. This study has revealed potential metabolic targets in response to the N status, which are indeed the prospective targets for crop improvement.

Roles of metabolic regulation in developing Quercus variabilis acorns at contrasting geologically-derived phosphorus sites in subtropical China

BackgroundPhosphorus (P) -rich soils develop in phosphorite residing areas while P-deficient soils are ubiquitous in subtropical regions. Little has been reported that how metabolites participate in the seed development and the processes involved in their coping with contrasting-nutrient environments.ResultsHere we quantified the metabolites of Quercus variabilis acorns in the early (July), middle (August), late (September) development stages, and determined element (C, H, O, N, P, K, Ca, Mg, S, Fe, Al, Mn, Na, Zn, and Cu) concentrations of acorns in the late stage, at geologically-derived contrasting-P sites in subtropical China. The primary metabolic pathways included sugar metabolism, the TCA cycle, and amino acid metabolism. Most metabolites (especially C- and N-containing metabolites) increased and then decreased from July to September. Acorns between the two sites were significantly discriminated at the three stages, respectively, by metabolites (predominantly sugars and organic acids). Concentrations of P, orthophosphoric acid and most sugars were higher; erythrose was lower in late-stage acorns at P-rich sites than those at P-deficient sites. No significant differences existed in the size and dry mass of individual acorns between oak populations at the two sites.ConclusionsOak acorns at the two sites formed distinct metabolic phenotypes related to their distinct geologically-derived soil conditions, and the late-stage acorns tended to increase P-use-efficiency in the material synthesis process at P-deficient sites, relative to those at P-rich sites.

Use of Untargeted Liquid Chromatography-Mass Spectrometry Metabolome To Discriminate Italian Monovarietal Red Wines, Produced in Their Different Terroirs.

The aim of this project was to register, in a liquid chromatography–mass spectrometry-based untargeted single-batch analysis, the metabolome of 11 single-cultivar, single-vintage Italian red wines (Aglianico, Cannonau, Corvina, Montepulciano, Nebbiolo, Nerello, Primitivo, Raboso, Sagrantino, Sangiovese, and Teroldego) from 12 regions across Italy, each one produced in their terroirs under ad hoc legal frameworks to guarantee their quality and origin. The data provided indications regarding the similarity between the cultivars and highlighted a rich list of putative biomarkers of origin wines (pBOWs) characterizing each individual cultivar–terroir combination, where Primitivo, Teroldego, and Nebbiolo had the maximum number of unique pBOWs. The pBOWs included anthocyanins (Teroldego), flavanols (Aglianico, Sangiovese, Nerello, and Nebbiolo), amino acids and N-containing metabolites (Primitivo), hydroxycinnamates (Cannonau), and flavonols (Sangiovese). The raw data generated in this study are publicly available and, therefore, accessible and reusable as a baseline data set for future investigations.

Metabolomics with 15N Labeling for Characterizing Missing Monoterpene Indole Alkaloids in Plants.

Monoterpene indole alkaloids (MIAs) in medicinal plants remain uncharacterized owing to their complicated structure by metabolomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) despite their pharmaceutical importance. We demonstrate an untargeted metabolome analysis with 15nitrogen (N) labeling to characterize MIAs having an indolic skeleton in the flowers, leaves, petioles, stems, and roots of Catharanthus roseus. Principal component analysis using 15N- and nonlabeled metabolome data showed that N-containing metabolites (N-metabolites) are labeled with 15N. Paring of the 15N- and nonlabeled precursor ions were performed using the criteria of retention time, difference of m/z value, and a nonlabeled product ion at m/z 144.08 that indicates an indolic skeleton. The mass shift of the m/z value of the product and precursor ions to their 15N-labeled ions identified the number of N of their ions. Finally, molecular formula of 45 MIAs was unambiguously identified using the identified N number. The alkaloid network analysis using the MS/MS similarity showed the structural commonness and uniqueness among the MIAs. Of them, antirhine was identified using an authentic standard compound. Multimetabolomics using LC-MS/MS and imaging mass spectrometry showed that antirhine accumulates considerably in the epidermis and vascular cylinder of the roots. The developed approach showed the existence of the missing MIAs. The modification of this approach will identify other MIAs that contain a hydroxylated or methoxylated indolic skeleton.

Metabolome and ionome analyses reveal the stoichiometric effects of contrasting geological phosphorus soils on seed-parasitic insects in subtropical oak forests

Phosphorus (P)-rich sites develop on phosphate-rock ores, while the soils are generally characterized with P deficiency in subtropical areas, resulting in contrasting nutrient environments for plants and herbivores. It remains unclear how in situ herbivorous insects cope with such two extreme nutrient habitats in terms of metabolome and ionome. Here, we investigated the metabolome and ionome of the weevil larvae (Curculio davidi Fairmaire), which were parasitizing in Quercus variabilis acorns at P-rich and P-deficient sites. Our results showed that there were significant differences in 34 identified metabolites (belonging to sugars, amino acids, lipids, vitamins, nucleosides, etc.) and four chemical elements (P, S, Mg, and Zn) in the two weevil larva populations of the two P-type sites. Moreover, the concentrations of P, Mg, Zn and the identified sugars were significantly higher; however, S, amino acids, and several other N-containing metabolites were lower in the weevil larvae at the P-rich site, in contrast to those at the P-deficient site. Arginine and proline metabolism and glutathione metabolism were the most relevant pathways differentially regulated between the two weevil larva populations at the two contrasting sites. In addition, some metabolites in the weevil larvae were indirectly associated with the P, Mg, Zn, and S concentrations of soils through bottom-up effects. Our results suggested that in situ herbivorous consumers altered their metabolites to a certain extent to adapt to nutrient-varying environments; and there were strong interactions between the nutrients of herbivorous insects and soil elements across variable nutrient sites.

Correlated metabolic and elemental variations between the leaves and seeds of oak trees at contrasting geologically derived phosphorus sites.

The leaves and seeds of plants frequently function as the source and sink organs for distinct metabolites, which can interactively vary in response to adverse site conditions. Subtropical soils are typically characterized as having deficient phosphorus (P), calcium (Ca), and magnesium (Mg), with enriched aluminum (Al) and iron (Fe), while Al and manganese (Mn) are toxic at low pH. It remains largely unknown how leaf- and seed-sourced metabolites are synergistically linked to adapt to P-variable soils for trees in subtropical areas. Here we quantified the metabolic and elemental profiling in the mature leaves and immature seeds of Quercus variabilis at contrasting geologically-derived phosphorus sites in subtropical China. The results revealed that carbon (C) and nitrogen (N) based metabolites (primarily sugars and organic acids), as well as enzyme- and protein/nucleic acid-related elements (N, P, Mg, and Mn) played important roles toward characterizing the profiling of metabolites and ionomes in leaves and seeds at two site types, respectively. These metabolites (sugars, amino acids, and fatty acids) and elements (N, P, Mg, and Mn) of seeds were closely related to the sugars, organic acids, and elements (N, P, Mg, and Mn) of leaves at the two site types. For the most part, the content of N and P in the soil affected the accumulation of materials (such as, starchs and proteins) in seeds, as well as N and P assimilation in leaves, by influencing C- and N-containing metabolites in leaves. These results suggested that correlated disparities of C- and N-containing metabolites, along with enzyme- and protein/nucleic acid-related elements in both leaves and seeds played important roles in plants to facilitate their adaptation to nutrient-variable sites in subtropical zones.

Chemometric analysis of Amaranthus retroflexus in relation to livestock toxicity in southern Australia

Amaranthus retroflexus L., an introduced invasive weed in southern Australia, has been associated with acute renal failure and/or mortality in a number of livestock species. While its leaves, flowers and stems are generally reported to contain high levels of nitrogen, few studies have fully characterised the chemical composition of A. retroflexus foliage with respect to mammalian toxicity. We performed extensive metabolic profiling of stems, leaves, roots and inflorescence tissues of A. retroflexus collected from three spatially and/or temporally distinct toxicity outbreaks, and report on the 1) composition of primary and secondary metabolites in methanolic extracts of A. retroflexus tissues using HPLC and HPLC-MS QToF and 2) chemometric analysis of A. retroflexus extracts in relation to the associated toxin(s). All tissues of A. retroflexus possessed an abundance of N-containing metabolites, particularly quaternary ammonium compounds which were identified as betaines, two of which (valine betaine and isoleucine betaine) are rarely encountered in plants. Cytotoxicity to murine fibroblasts was highest in extracts of leaf tissue and was associated with a single, a small modified peptide with high similarity to N-acetyl-L-α-aspartyl-L-alanyl-L-α-aspartyl-L-α-glutamyl-O-(carboxymethyl)-L-tyrosyl-L-leucinamide, a synthetic phosphotyrosyl mimic involved in cell signaling processes. One possible mode of action leading to acute renal failure in grazing livestock by a modified peptide such as this is proposed.

Primed primary metabolism in systemic leaves: a functional systems analysis

Plants evolved mechanisms to counteract bacterial infection by preparing yet uninfected systemic tissues for an enhanced defense response, so-called systemic acquired resistance or priming responses. Primed leaves express a wide range of genes that enhance the defense response once an infection takes place. While hormone-driven defense signalling and defensive metabolites have been well studied, less focus has been set on the reorganization of primary metabolism in systemic leaves. Since primary metabolism plays an essential role during defense to provide energy and chemical building blocks, we investigated changes in primary metabolism at RNA and metabolite levels in systemic leaves of Arabidopsis thaliana plants that were locally infected with Pseudomonas syringae. Known defense genes were still activated 3–4 days after infection. Also primary metabolism was significantly altered. Nitrogen (N)-metabolism and content of amino acids and other N-containing metabolites were significantly reduced, whereas the organic acids fumarate and malate were strongly increased. We suggest that reduction of N-metabolites in systemic leaves primes defense against bacterial infection by reducing the nutritional value of systemic tissue. Increased organic acids serve as quickly available metabolic resources of energy and carbon-building blocks for the production of defense metabolites during subsequent secondary infections.

Tracing the Biotransformation of Polycyclic Aromatic Hydrocarbons in Contaminated Soil Using Stable Isotope-Assisted Metabolomics.

Biotransformation of organic pollutants may result in the formation of oxidation products more toxic than the parent contaminants. However, to trace and identify those products, and the metabolic pathways involved in their formation, is still challenging within complex environmental samples. We applied stable isotope-assisted metabolomics (SIAM) to PAH-contaminated soil collected from a wood treatment facility. Soil samples were separately spiked with uniformly 13C-labeled fluoranthene, pyrene, or benzo[a]anthracene at a level below that of the native contaminant, and incubated for 1 or 2 weeks under aerobic biostimulated conditions. Combining high-resolution mass spectrometry and automated SIAM workflows, chemical structures of metabolites and metabolic pathways in the soil were proposed. Ring-cleavage products, including previously unreported intermediates such as C11H10O6 and C15H12O5, were detected originating from fluoranthene and benzo[a]anthracene, respectively. Sulfate conjugates of dihydroxy compounds were found as major metabolites of pyrene and benzo[a]anthracene, suggesting the potential role of fungi in their biotransformation in soils. A series of unknown N-containing metabolites were identified from pyrene, but their structural elucidation requires further investigation. Our results suggest that SIAM can be successfully applied to understand the fate of organic pollutants in environmental samples, opening lines of evidence for novel mechanisms of microbial transformation within such complex matrices.

Simultaneous efflux and uptake of metabolites by roots of wheat

Some metabolites (e.g. amino acids) present in root exudates can be taken up by roots, but we do not know if this ability extends to the broader suite of metabolites found in exudates. The aim of this study was to examine the ability of wheat (Triticum aestivum L.) to efflux and take up a broad suite of small metabolites. Four-week-old plants were placed into an uptake solution that contained a broad suite of 13C-labelled metabolites. Uptake was estimated from the decrease in concentration of the 13C isotopologue in the solution; and from the appearance of 13C isotopologues within roots. Efflux was estimated from appearance of 12C isotopologues in solution. When wheat plants were placed in 13C-metabolite solutions the concentration of U-13C isotopologues of 37 metabolites decreased – as would be expected if plants were taking up the U-13C metabolites. After 4 h immersion in 13C metabolite solution, roots contained detectable amounts of U-13C isotopologues of 55 metabolites. U-13C isotopologues of organic acids were not detected within roots. These findings indicate that wheat can take up a broad suite of N-containing metabolites and some sugars, but there was no evidence for uptake of organic acids.

Induced defense mechanisms in an aquatic angiosperm to insect herbivory

In terrestrial angiosperms, defense and resistance mechanisms against herbivores have been studied extensively; yet this topic is poorly understood in aquatic angiosperms. We investigated induced response mechanisms in Myriophyllum spicatum to the generalist insect herbivore Acentria ephemerella in three independent experiments. Various morphological and chemical response variables were examined in grazed apical shoots and compared to undamaged controls. We further estimated plant palatability of induced and non-induced apices in choice assays, and assessed the growth response of Acentria larvae in no-choice feeding assays. Leaves of induced apices were splayed out horizontally and changed in color from green to red. The dry matter content and thus plant toughness increased by up to 19%, but silica levels stayed constant. Induced apices exhibited a decline in chlorophyll content of up to 34%, reflected also by a 10% decrease in nitrogen levels, while nitrogen increased by 14% in lower parts of grazed shoots. Also, herbivore-deterring total phenolic compounds increased by up to 20% in apices. In choice trials, Acentria larvae strongly avoided grazed tips, and growth was reduced by 25% on induced apices. In total, we observed five different induced resistance and defensive traits in grazed apices: changes in appearance, increased plant toughness, delocalization of N-containing metabolites, increased polyphenols, and reduced nutritional value. The observed changes prevent herbivore damage and loss of apical tissue, which are most valuable for plant fitness. Our study presents the first evidence of multiple, parallel defense strategies including constitutive and induced defense mechanisms in a freshwater angiosperm.

N-Containing Metabolites from the Marine Sponge Agelas clathrodes

A new bisuracil analogue, 3,3-bis(uracil-1-yl)-propan-1-aminium (1), together with four known N-containing metabolites (2-5), were isolated from the South China Sea sponge Agelas clathrodes. Their chemical structures were established on the basis of spectroscopic and spectrometric analysis and comparison with known compounds. Compound 1 is an unusual naturally occurring bisuracil analogue, and compound 2 was isolated from a natural source for the first time. Compounds 2 and 4 exhibit moderate cytotoxicity against cancer cell line SGC7901.