A Novel Signaling Pathway Required for Arabidopsis Endodermal Root Organization Shapes the Rhizosphere Microbiome.

The Casparian strip (CS) constitutes a physical diffusion barrier to water and nutrients in plant roots, which is formed by the polar deposition of lignin polymer in the endodermis tissue. The precise pattern of lignin deposition is determined by the scaffolding activity of membrane-bound Casparian Strip domain proteins (CASPs), but little is known of the mechanism(s) directing this process. Here, we demonstrate that Endodermis-specific Receptor-like Kinase 1 (ERK1) and, to a lesser extent, ROP Binding Kinase1 (RBK1) are also involved in regulating CS formation, with the former playing an essential role in lignin deposition as well as in the localization of CASP1. We show that ERK1 is localized to the cytoplasm and nucleus of the endodermis and that together with the circadian clock regulator, Time for Coffee (TIC), forms part of a novel signaling pathway necessary for correct CS organization and suberization of the endodermis, with their single or combined loss of function resulting in altered root microbiome composition. In addition, we found that other mutants displaying defects in suberin deposition at the CS also display altered root exudates and microbiome composition. Thus, our work reveals a complex network of signaling factors operating within the root endodermis that establish both the CS diffusion barrier and influence the microbial composition of the rhizosphere.

Hybrid Genome Assembly for Predicting Functional Potential of a Novel Streptomyces Strain as Plant Biomass Valorisation Agent.

Environmental bioremediation relies heavily on the realized potential of efficient bioremediation agents or microbial strains of interest. Identifying suitable microbial agents for plant biomass waste valorization requires (i) high-quality genome assemblies to predict the full metabolic and functional potential, (ii) accurate mapping of lignocellulose metabolizing enzymes. However, fragmented nature of the sequenced genomes often limits the prediction ability due to breaks occurring in coding sequences. To address these challenges and as part of our ongoing agri-culturomics efforts, we have performed a hybrid genome assembly using Illumina and Nanopore reads with modified assembly protocol, for a novel Streptomyces strain isolated from the rhizosphere niche of green leafy vegetables grown in a commercial urban farm. High-quality genome was assembled with the size of 8.6 Mb in just two contigs with N50 of 8,542,030 and coverage of 383X. This facilitated identification and complete arrangement of approximately 248 CAZymes and 38 biosynthetic gene clusters in the genome. Multiple gene clusters consisting of cellulases and hemicellulases associated with substrate recognition domain were identified in the genome. Genes for lignin, chitin, and even some aromatic compounds degradation were found in the Streptomyces sp. genome which makes it a promising candidate for lignocellulosic waste valorization. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00935-5.

Insights into lipidomic perturbations in zebrafish tissues upon exposure to microcystin-LR and microcystin-RR.

This work represents the first study of its kind that was conducted to evaluate changes in lipid metabolic networks following a balneation exposure of adult zebrafish to MCLR (microcystin-leucine-arginine) and MCRR (microcystin-arginine-arginine) at a sublethal dose (10 µg L(-1)) for a period of 30 days. Following the exposure to MCLR and MCRR, gills, liver, intestine, and brain tissues were harvested for metabolite extraction. Extracted metabolites were detected using qTOF-LC-MS (time-of-flight-liquid chromatography-mass spectrometry). Metabolites were identified using Kegg pathways. The identified metabolites are shown on lipid biochemical maps to demonstrate major perturbations in the metabolic machinery. Results showed that most of the metabolic pathways under the lipid class were affected in different tissues of zebrafish following the exposure to MCLR and MCRR (10 µg L(-1) for 30 days). The kind and flux of metabolic perturbations varied among different tissues of the organs after the exposure to MCLR and MCRR with the tissues of gills being the most affected. Among the various lipid pathways, cholesterol synthesis was affected significantly as observed from the highest number of perturbed metabolites in that pathway. Cholesterol is responsible for synthesis of steroid hormones and bile acids, which have been recognized as endocrine signaling molecules. Disruption in the synthesis of these compounds following MCLR/MCRR exposure suggests that MCs are capable of causing endocrine disruption among aquatic organisms even under sublethal conditions. Apart from cholesterol synthesis, various other metabolic pathways belonging to the class of essential fatty acids and lipid oxidation were also observed to be perturbed following a balneation exposure of zebrafish to MCLR/MCRR.

Global analyses of biosynthetic gene clusters in phytobiomes reveal strong phylogenetic conservation of terpenes and aryl polyenes.

There are gaps in our understandings on how did the evolutionary relationships among members of the phytobiomes shape their ability to produce tremendously complex specialized metabolites under the influence of plant host. To determine these relationships, we investigated the phylogenetic conservation of biosynthetic gene clusters (BGCs) on a global collection of 4,519 high-quality and nonredundant (out of 12,181) bacterial isolates and metagenome-assembled genomes from 47 different plant hosts and soil, by adopting three independent phylogenomic approaches (D-test, Pagel's λ, and consenTRAIT). We report that the BGCs are phylogenetically conserved to varying strengths and depths in their different classes. We show that the ability to produce specialized metabolites qualifies as a complex trait, and the depth of conservation is equivalent to ecologically relevant complex microbial traits. Interestingly, terpene and aryl polyene BGCs had the strongest phylogenetic conservation in the phytobiomes, but not in the soil microbiomes. Furthermore, we showed that terpenes are largely uncharacterized in phytobiomes and pinpointed specific clades that harbor potentially novel terpenes. Taken together, this study sheds light on the evolution of specialized metabolites' biosynthesis potential in phytobiomes under the influence of plant hosts and presents strategies to rationally guide the discovery of potentially novel classes of metabolites. IMPORTANCE This study expands our understandings of the biosynthetic potential of phytobiomes by using such worldwide and extensive collection of microbiomes from plants and soil. Apart from providing such vital resource for the plant microbiome researchers, this study provides fundamental insights into the evolution of biosynthetic gene clusters (BGCs) in phytobiomes under the influence of plant host. Specifically, we report that the strength of phylogenetic conservation in microbiomes varies for different classes of BGCs and is influenced as a result of plant host association. Furthermore, our results indicate that biosynthetic potential of specialized metabolites is deeply conserved equivalent to other complex and ecologically relevant microbial traits. Finally, for the most conserved class of specialized metabolites (terpenes), we identified clades harboring potentially novel class of molecules. Future studies could focus on plant-microbe coevolution and interactions through specialized metabolites building upon these findings.

Penicillium citrinum Provides Transkingdom Growth Benefits in Choy Sum (Brassica rapa var. parachinensis).

Soil-borne beneficial microbes establish symbioses with plant hosts and play key roles during growth and development therein. In this study, two fungal strains, FLP7 and B9, were isolated from the rhizosphere microbiome associated with Choy Sum (Brassica rapa var. parachinensis) and barley (Hordeum vulgare), respectively. Sequence analyses of the internal transcribed spacer and 18S ribosomal RNA genes combined with colony and conidial morphology identified FLP7 and B9 to be Penicillium citrinum strains/isolates. Plant-fungus interaction assays revealed that isolate B9 showed significant growth promotion effects in Choy Sum plants cultivated in normal soil, as well as under phosphate-limiting conditions. In comparison to the mock control, B9-inoculated plants showed a 34% increase in growth in aerial parts and an 85% upsurge in the fresh weight of roots when cultivated in sterilized soil. The dry biomass of such fungus-inoculated Choy Sum increased by 39% and 74% for the shoots and roots, respectively. Root colonization assays showed that P. citrinum associates directly with the root surface but does not enter or invade the root cortex of the inoculated Choy Sum plants. Preliminary results also indicated that P. citrinum can promote growth in Choy Sum via volatile metabolites too. Interestingly, we detected relatively higher amounts of gibberellins and cytokinins in axenic P. citrinum culture filtrates through liquid chromatography-mass spectrometry analyses. This could plausibly explain the overall growth induction in P. citrinum-inoculated Choy Sum plants. Furthermore, the phenotypic growth defects associated with the Arabidopsis ga1 mutant could be chemically complemented by the exogenous application of P. citrinum culture filtrate, which also showed accumulation of fungus-derived active gibberellins. Our study underscores the importance of transkingdom beneficial effects of such mycobiome-assisted nutrient assimilation and beneficial fungus-derived phytohormone-like metabolites in the induction of robust growth in urban farmed crops.

Effects of Light and Temperature on the Metabolic Profiling of Two Habitat-Dependent Bloom-Forming Cyanobacteria.

Rapid proliferation of cyanobacteria in both benthic and suspended (planktonic) habitats is a major threat to environmental safety, as they produce nuisance compounds such as cytotoxins and off-flavors, which degrade the safety and quality of water supplies. Temperature and light irradiance are two of the key factors in regulating the occurrence of algal blooms and production of major off-flavors. However, the role of these factors in regulating the growth and metabolism is poorly explored for both benthic and planktonic cyanobacteria. To fill this gap, we studied the effects of light and temperature on the growth and metabolic profiling of both benthic (Hapalosiphon sp. MRB220) and planktonic (Planktothricoides sp. SR001) environmental species collected from a freshwater reservoir in Singapore. Moreover, this study is the first report on the metabolic profiling of cyanobacteria belonging to two different habitats in response to altered environmental conditions. The highest growth rate of both species was observed at the highest light intensity (100 µmol photons/m²/s) and at a temperature of 33 °C. Systematic metabolite profiling analysis suggested that temperature had a more profound effect on metabolome of the Hapalosiphon, whereas light had a greater effect in the case of Planktothricoides. Interestingly, Planktothricoides sp. SR001 showed a specialized adaptation mechanism via biosynthesis of arginine, and metabolism of cysteine and methionine to survive and withstand higher temperatures of 38 °C and higher. Hence, the mode of strategies for coping with different light and temperature conditions was correlated with the growth and alteration in metabolic activities for physiological and ecological adaptations in both species. In addition, we putatively identified a number of unique metabolites with a broad range of antimicrobial activities in both species in response to both light and temperature. These metabolites could play a role in the dominant behavior of these species in suppressing competition during bloom formation. Overall, this study elucidated novel insights into the effects of environmental factors on the growth, metabolism, and adaptation strategies of cyanobacteria from two different habitats, and could be useful in controlling their harmful effects on human health and environmental concerns.

Metabolomics for Evaluating Flavor-Associated Metabolites in Plant-Based Products.

Plant-based diets (PBDs) are associated with environmental benefits, human health promotion and animal welfare. There is a worldwide shift towards PBDs, evident from the increased global demand for fresh plant-based products (PBPs). Such shifts in dietary preferences accompanied by evolving food palates, create opportunities to leverage technological advancements and strict quality controls in developing PBPs that can drive consumer acceptance. Flavor, color and texture are important sensory attributes of a food product and, have the largest influence on consumer appeal and acceptance. Among these, flavor is considered the most dominating quality attribute that significantly affects overall eating experience. Current state-of-art technologies rely on physicochemical estimations and sensory-based tests to assess flavor-related attributes in fresh PBPs. However, these methodologies often do not provide any indication about the metabolic features associated with unique flavor profiles and, consequently, can be used in a limited way to define the quality attributes of PBPs. To this end, a systematic understanding of metabolites that contribute to the flavor profiles of PBPs is warranted to complement the existing methodologies. This review will discuss the use of metabolomics for evaluating flavor-associated metabolites in fresh PBPs at post-harvest stage, alongside its applications for quality assessment and grading. We will summarize the current research in this area, discuss technical challenges and considerations pertaining to sampling and analytical techniques, as well as s provide future perspectives and directions for government organizations, industries and other stakeholders associated with the quality assessment of fresh PBPs.

910 metagenome-assembled genomes from the phytobiomes of three urban-farmed leafy Asian greens.

The genome sequences of many microbial species from the phytobiomes of several leafy Asian greens remain unknown. Here, we address this gap by reconstructing 910 prokaryotic draft genomes from 24 leaf, 65 root, 12 soil, and 6 compost metagenomes from the seedling and adult developmental stages of three leafy Asian greens - Brassica rapa var. parachinensis, Brassica oleracea var. alboglabra and Amaranthus spp. - grown in a commercial, soil-based urban farm. Of these, 128 are near-complete (>90% completeness, <5% redundancy), 540 are substantially complete (≥70% completeness, <10%, redundancy), while the rest have a completeness ≥50% and redundancy <10%. The draft genomes together span 292 bacterial and 3 archaeal species, a subset of which are from underrepresented genus-level lineages in public databases. We expect our dataset to facilitate a wide range of comparative studies that seek to understand the different functional aspects of vegetable crop phytobiomes and for devising new strategies for microbial cultivation in the future.

Extracellular Electron Transfer Powers Enterococcus faecalis Biofilm Metabolism.

Enterococci are important human commensals and significant opportunistic pathogens. Biofilm-related enterococcal infections, such as endocarditis, urinary tract infections, wound and surgical site infections, and medical device-associated infections, often become chronic upon the formation of biofilm. The biofilm matrix establishes properties that distinguish this state from free-living bacterial cells and increase tolerance to antimicrobial interventions. The metabolic versatility of the enterococci is reflected in the diversity and complexity of environments and communities in which they thrive. Understanding metabolic factors governing colonization and persistence in different host niches can reveal factors influencing the transition to biofilm pathogenicity. Here, we report a form of iron-dependent metabolism for Enterococcus faecalis where, in the absence of heme, extracellular electron transfer (EET) and increased ATP production augment biofilm growth. We observe alterations in biofilm matrix depth and composition during iron-augmented biofilm growth. We show that the ldh gene encoding l-lactate dehydrogenase is required for iron-augmented energy production and biofilm formation and promotes EET.IMPORTANCE Bacterial metabolic versatility can often influence the outcome of host-pathogen interactions, yet causes of metabolic shifts are difficult to resolve. The bacterial biofilm matrix provides the structural and functional support that distinguishes this state from free-living bacterial cells. Here, we show that the biofilm matrix can immobilize iron, providing access to this growth-promoting resource which is otherwise inaccessible in the planktonic state. Our data show that in the absence of heme, Enterococcus faecalis l-lactate dehydrogenase promotes EET and uses matrix-associated iron to carry out EET. Therefore, the presence of iron within the biofilm matrix leads to enhanced biofilm growth.

Uptake of Ag and TiO2 nanoparticles by zebrafish embryos in the presence of other contaminants in the aquatic environment.

The present study aimed at evaluating the uptake of two widely used nanoparticles (NPs), namely, silver (Ag) and titanium dioxide (TiO2) NPs by zebrafish embryos under different simulated environmental conditions. AgNPs and TiO2NPs are widely used in a number of consumer products, and are thus likely to be introduced into the aquatic environments from both domestic and industrial sources. These NPs could interact with other contaminants in aquatic systems, which could affect their uptake by fish and biodistribution kinetics. To provide insights into these interactions, uptake studies were conducted in the presence of 12 elements and 3 major inorganic nutrients using (1) single (AgNPs and TiO2NPs separately), (2) binary (containing either both AgNPs and TiO2NPs or one of the NPs and elements/nutrients) and (3) multi-components (conducted in real water samples collected from a tropical reservoir). In addition to the uptake of NPs, mortality, hatchability and heart rates of zebrafish embryos were also measured as part of these experiments. Results showed that AgNPs were taken up by the embryos to a larger extent as compared to TiO2NPs. Moreover, AgNPs were more toxic to zebrafish embryos as compared to TiO2NPs. In the presence of elements (5 µg mL(-1)) and nutrients (5 and 10 µg mL(-1)) together with one of the two NPs (10 µg mL(-1)), both an increase and a decrease in the uptake of NPs were observed in embryos. Results from this exploratory study show that in the presence of environmental contaminants, the uptake of these NPs can be affected significantly. Furthermore, the toxic effects of NPs depend not only on their relative environmental concentrations, but also on those of other environmental pollutants.

Toxicological evaluation of microcystins in aquatic fish species: current knowledge and future directions.

Microcystins (MCs) are algal toxins produced intracellularly within the algal cells, and are subsequently released into the aquatic systems. An increase in the frequency and intensity of occurrence of harmful algal blooms has directed the global attention towards the presence of MCs in aquatic systems. The effects of MCs on fish have been verified in a number of studies including histological, biochemical and behavioral effects. The toxicological effects of MCs on different organs of fish are related to the exposure route (intraperitoneal injection, feeding or immersion), the mode of uptake (passive or active transport) as well as biotransformation and bioaccumulation capabilities by different organs. This paper reviews the rapidly expanding literature on the toxicological evaluation of MCs in fish from both field studies and controlled laboratory experimental investigations, integrates the current knowledge available about the mechanisms involved in MC-induced effects on fish, and points out future research directions from a cross-disciplinary perspective. In addition, the need to carry out systematic fish toxicity studies to account for possible interactions between MCs and other environmental pollutants in aquatic systems is discussed.

Toxicological implications of microcystins for zebrafish embryos in the presence of other environmental pollutants.

Microcystins (MCs) interact with environmental contaminants as well as various other congeners of the MC family in the natural environment and with antioxidants in the exposed organisms. These interactions are likely to modify the toxicological behavior of MCs at the cellular level. The present study was conducted to determine the toxicological response of extracellular MCs in aquatic systems under environmentally relevant conditions. Microcystin-leucine-arginine (MCLR) and microcystin-arginine-arginine (MCRR) were introduced at different concentrations in a single-component (MCLR or MCRR) or dual-component (MCLR and MCRR) system to zebrafish embryos in the presence of inorganic elements (Hg, As, Pb, and Cd) and nutrient species (NO3 (-) , PO4 (3-) , and Cl(-1) ). Hatchability, heart rate, and mortality of zerbrafish embryos were monitored together with changes in the activity of glutathione-S-transferase (GST) to evaluate their response on exposure to MCLR and MCRR. There was a significant reduction in all these parameters at higher doses of MCLR and MCRR (>100 ng/mL), implying bioaccumulation of these MCs in embryos and adverse effects on early development stages of the fish. It was further observed that PO4 (3-) and Cl(-) enhanced the toxic effects of MCLR and MCRR while NO3 (-) attenuated their toxic effects. In contrast, all 4 toxic elements together increased the toxicity of MCLR and MCRR to embryos compared with their single-component counterparts. Thus, the toxic effects of MCs depend not only on their relative environmental concentrations, but also on those of other environmental pollutants and the levels of antioxidants in exposed organisms.

Removal of microcystin-LR and microcystin-RR by graphene oxide: adsorption and kinetic experiments.

Graphene oxide (GO) was employed in the present study for removal of two commonly occurring algal toxins, microcystin-LR (MC-LR) and microcystin-RR (MC-RR), from water. The adsorption performance of GO was compared to that of commercially available activated carbon. Further, adsorption experiments were conducted in the presence of other environmental pollutants to understand the matrix effects of contaminated water on the selective adsorption of MC-LR and MC-RR onto GO. The environmental pollutants addressed in this study included different anions (nitrate NO3-, nitrite NO2-, sulphate SO4(2-), chloride (Cl(-)), phosphate PO4(3-) and fluoride (F(-))) and cations (sodium (Na(+)), potassium (K(+)), magnesium (Mg(2+)) and calcium (Ca(2+))). GO showed very a high adsorption capacity of 1700 µg/g for removal of MC-LR and 1878 µg/g for MC-RR while the maximum adsorption capacity obtained with the commercial activated carbon was 1481.7 µg/g and 1034.1 µg/g for MC-LR and MC-RR, respectively. The sorption kinetic experiments revealed that more than 90% removal of both MC-LR/RR was achieved within 5 min for all the doses studied (500, 700 and 900 µg/L). GO could be reused as an adsorbent following ten cycles of adsorption/desorption with no significant loss in its adsorption capacity.

Physicochemical and toxicological characteristics of urban aerosols during a recent Indonesian biomass burning episode.

Air particulate matter (PM) samples were collected in Singapore from 21 to 29 October 2010. During this time period, a severe regional smoke haze episode lasted for a few days (21-23 October). Physicochemical and toxicological characteristics of both haze and non-haze aerosols were evaluated. The average mass concentration of PM2.5 (PM with aerodynamic diameter of ≤2.5 µm) increased by a factor of 4 during the smoke haze period (107.2 µg/m(3)) as compared to that during the non-smoke haze period (27.0 µg/m(3)). The PM2.5 samples were analyzed for 16 priority polycyclic aromatic hydrocarbons (PAHs) listed by the United States Environmental Protection Agency and 10 transition metals. Out of the seven PAHs known as potential or suspected carcinogens, five were found in significantly higher levels in smoke haze aerosols as compared to those in the background air. Metal concentrations were also found to be higher in haze aerosols. Additionally, the toxicological profile of the PM2.5 samples was evaluated using a human epithelial lung cell line (A549). Cell viability and death counts were measured after a direct exposure of PM2.5 samples to A459 cells for a period of 48 h. The percentage of metabolically active cells decreased significantly following a direct exposure to PM samples collected during the haze period. To provide further insights into the toxicological characteristics of the aerosol particles, glutathione levels, as an indirect measure of oxidative stress and caspase-3/7 levels as a measure of apoptotic death, were also evaluated.

Biochemical response of diverse organs in adult Danio rerio (zebrafish) exposed to sub-lethal concentrations of microcystin-LR and microcystin-RR: a balneation study.

The present study was carried out to examine the dose-response of microcystin-LR (MC-LR) and microcystin-RR (MC-RR) toxicity in adult Danio rerio (zebrafish) under balneation conditions at various time points. The differential responses of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST) as biomarkers were assessed for oxygen mediated toxicity in liver, gills, intestine and brain tissues of zebrafish exposed to dissolved MC-LR and MC-RR (0.1-10.0 µgl(-1)). To investigate the time related response of biomarkers, fish were sampled after 4, 7 and 15 days of exposure. Responses varied (i) between MC-LR and MC-RR (for certain groups), (ii) for different enzymes at all time points, and (iii) for different tissues. In general, most of the enzymes followed a bell shaped curve, with an abrupt increase in activity at a particular concentration. It was observed that upon exposure to MC-LR and MC-RR, some enzymes showed an adaptive response after the first time point wherein the enzyme activity increased in some tissues. The increase in enzyme activity is suggestive of their cellular and metabolic adaptations to the continued stress and toxin exposure. Enzyme activities in general increased at lower concentrations (≤ 5.0 µgl(-1)) and decreased at higher concentrations (≥ 5.0 µgl(-1)). An abrupt change in enzyme activities was observed at a particular concentration in all the tissue enzymes. For GPx and GR, there was a differential response in the case of fish exposed to MC-LR and MC-RR, which could be due to the difference in toxicity potentials of these cyanotoxins. In general, initial stress conditions were observed in most of the tissue enzymes following the exposure to microcystins (MCs). This observation suggests that MCs found in trace levels are likely to have deleterious effects on aquatic organisms and can trigger a variety of biochemical responses depending on their specific toxicity.

Application of ionic-liquid supported cloud point extraction for the determination of microcystin-leucine-arginine in natural waters.

A cloud point extraction method has been developed using an ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate, BMiM PF(6)) for the determination of a widely studied cyanotoxin (microcystin leucine-arginine, MCLR) in natural waters. Extraction parameters such as sample pH, extraction temperature, extraction time, the amount of ionic liquid and the amount of extraction volume were investigated and optimized to achieve the maximum extraction efficiency. The results obtained indicated a good linearity with the correlation coefficient of 0.995 over the range of 0.5-50 µg L(-1). The relative standard deviation (RSD) of the method was 7.5% (n=6). The calculated method detection limit was 0.03 µg L(-1) (n=6). The practical applicability of the technique was demonstrated by analyzing water samples (n=9) collected from three different sites in local reservoirs.

Determination of aldehydes in rainwater using micro-solid-phase extraction and high-performance liquid chromatography.

A simple and rapid extraction procedure was developed for determining aldehydes in rainwater samples. This extraction technique involved the use of micro-solid-phase extraction in which the sorbent was held within a polypropylene membrane envelope, followed by high-performance liquid chromatographic analysis. Aldehydes such as formaldehyde, acetaldehyde, propionaldehyde and valeraldehyde were used as model compounds. Extraction conditions were optimized. The method linearity ranged between 0.5 and 50 µgl(-1) with the correlation coefficient of 0.987-0.999. The relative standard deviations (RSDs) of the method ranged from 7 to 12%. Method detection limits were in the range of 0.07-0.15 µgl(-1), which is lower than those previously reported for solid-phase microextraction combined with gas chromatography-mass spectrometric techniques. The proposed extraction technique was used for determination of aldehydes in rainwater samples to demonstrate the applicability of the method.