Root-exuded specialized metabolites reduce arsenic toxicity in maize.

By releasing specialized metabolites, plants modify their environment. Whether and how specialized metabolites protect plants against toxic levels of trace elements is not well understood. We evaluated whether benzoxazinoids, which are released into the soil by major cereals, can confer protection against arsenic toxicity. Benzoxazinoid-producing maize plants performed better in arsenic-contaminated soils than benzoxazinoid-deficient mutants in the greenhouse and the field. Adding benzoxazinoids to the soil restored the protective effect, and the effect persisted to the next crop generation via positive plant-soil feedback. Arsenate levels in the soil and total arsenic levels in the roots were lower in the presence of benzoxazinoids. Thus, the protective effect of benzoxazinoids is likely soil-mediated and includes changes in soil arsenic speciation and root accumulation. We conclude that exuded specialized metabolites can enhance protection against toxic trace elements via soil-mediated processes and may thereby stabilize crop productivity in polluted agroecosystems.

Soil chemistry determines whether defensive plant secondary metabolites promote or suppress herbivore growth.

Plant secondary (or specialized) metabolites mediate important interactions in both the rhizosphere and the phyllosphere. If and how such compartmentalized functions interact to determine plant-environment interactions is not well understood. Here, we investigated how the dual role of maize benzoxazinoids as leaf defenses and root siderophores shapes the interaction between maize and a major global insect pest, the fall armyworm. We find that benzoxazinoids suppress fall armyworm growth when plants are grown in soils with very low available iron but enhance growth in soils with higher available iron. Manipulation experiments confirm that benzoxazinoids suppress herbivore growth under iron-deficient conditions and in the presence of chelated iron but enhance herbivore growth in the presence of free iron in the growth medium. This reversal of the protective effect of benzoxazinoids is not associated with major changes in plant primary metabolism. Plant defense activation is modulated by the interplay between soil iron and benzoxazinoids but does not explain fall armyworm performance. Instead, increased iron supply to the fall armyworm by benzoxazinoids in the presence of free iron enhances larval performance. This work identifies soil chemistry as a decisive factor for the impact of plant secondary metabolites on herbivore growth. It also demonstrates how the multifunctionality of plant secondary metabolites drives interactions between abiotic and biotic factors, with potential consequences for plant resistance in variable environments.

Variability in Arsenic Methylation Efficiency across Aerobic and Anaerobic Microorganisms.

Microbially-mediated methylation of arsenic (As) plays an important role in the As biogeochemical cycle, particularly in rice paddy soils where methylated As, generated microbially, is translocated into rice grains. The presence of the arsenite (As(III)) methyltransferase gene (arsM) in soil microbes has been used as an indication of their capacity for As methylation. Here, we evaluate the ability of seven microorganisms encoding active ArsM enzymes to methylate As. Amongst those, only the aerobic species were efficient methylators. The anaerobic microorganisms presented high resistance to As exposure, presumably through their efficient As(III) efflux, but methylated As poorly. The only exception were methanogens, for which efficient As methylation was seemingly an artifact of membrane disruption. Deletion of an efflux pump gene (acr3) in one of the anaerobes, Clostridium pasteurianum, rendered the strain sensitive to As and capable of more efficiently methylating As. Our results led to the following conclusions: (i) encoding a functional ArsM enzyme does not guarantee that a microorganism will actively drive As methylation in the presence of the metalloid and (ii) there is an inverse relationship between efficient microbial As efflux and its methylation, because the former prevents the intracellular accumulation of As.

A Herbivore Tag-and-Trace System Reveals Contact- and Density-Dependent Repellence of a Root Toxin.

Foraging behavior of root feeding organisms strongly affects plant-environment-interactions and ecosystem processes. However, the impact of plant chemistry on root herbivore movement in the soil is poorly understood. Here, we apply a simple technique to trace the movement of soil-dwelling insects in their habitats without disturbing or restricting their interactions with host plants. We tagged the root feeding larvae of Melolontha melolontha with a copper ring and repeatedly located their position in relation to their preferred host plant, Taraxacum officinale, using a commercial metal detector. This method was validated and used to study the influence of the sesquiterpene lactone taraxinic acid ß-D-glucopyranosyl ester (TA-G) on the foraging of M. melolontha. TA-G is stored in the latex of T. officinale and protects the roots from herbivory. Using behavioral arenas with TA-G deficient and control plants, we tested the impact of physical root access and plant distance on the effect of TA-G on M. melolontha. The larvae preferred TA-G deficient plants to control plants, but only when physical root contact was possible and the plants were separated by 5 cm. Melolontha melolontha showed no preference for TA-G deficient plants when the plants were grown 15 cm apart, which may indicate a trade-off between the cost of movement and the benefit of consuming less toxic food. We demonstrate that M. melolontha integrates host plant quality and distance into its foraging patterns and suggest that plant chemistry affects root herbivore behavior in a plant-density dependent manner.

Identification and quantification of phytochelatins in roots of rice to long-term exposure: evidence of individual role on arsenic accumulation and translocation.

Rice has the predilection to take up arsenic in the form of methylated arsenic (o-As) and inorganic arsenic species (i-As). Plants defend themselves using i-As efflux systems and the production of phytochelatins (PCs) to complex i-As. Our study focused on the identification and quantification of phytochelatins by HPLC-ICP-MS/ESI-MS, relating them to the several variables linked to As exposure. GSH, 11 PCs, and As-PC complexes from the roots of six rice cultivars (Italica Carolina, Dom Sofid, 9524, Kitrana 508, YRL-1, and Lemont) exposed to low and high levels of i-As were compared with total, i-As, and o-As in roots, shoots, and grains. Only Dom Sofid, Kitrana 508, and 9524 were found to produce higher levels of PCs even when exposed to low levels of As. PCs were only correlated to i-As in the roots (r=0.884, P <0.001). However, significant negative correlations to As transfer factors (TF) roots-grains (r= -0.739, P <0.05) and shoots-grains (r= -0.541, P <0.05), suggested that these peptides help in trapping i-As but not o-As in the roots, reducing grains' i-As. Italica Carolina reduced i-As in grains after high exposure, where some specific PCs had a special role in this reduction. In Lemont, exposure to elevated levels of i-As did not result in higher i-As levels in the grains and there were no significant increases in PCs or thiols. Finally, the high production of PCs in Kitrana 508 and Dom Sofid in response to high As treatment did not relate to a reduction of i-As in grains, suggesting that other mechanisms such as As-PC release and transport seems to be important in determining grain As in these cultivars.

Arsenic speciation and localization in horticultural produce grown in a historically impacted mining region.

A field and market basket study (~1300 samples) of locally grown fruits and vegetables from historically mined regions of southwest (SW) England (Cornwall and Devon), and as reference, a market basket study of similarly locally grown produce from the northeast (NE) of Scotland (Aberdeenshire) was conducted to determine the concentration of total and inorganic arsenic present in produce from these two geogenically different areas of the U.K. On average 98.5% of the total arsenic found was present in the inorganic form. For both the market basket and the field survey, the highest total arsenic was present in open leaf structure produce (i.e., kale, chard, lettuce, greens, and spinach) being most likely to soil/dust contamination of the open leaf structure. The concentration of total arsenic in potatoes, swedes, and carrots was lower in peeled produce compared to unpeeled produce. For baked potatoes, the concentration of total arsenic in the skin was higher compared to the total arsenic concentration of the potato flesh, this difference in localization being confirmed by laser ablation inductively coupled plasma mass spectroscopy (LA-ICP-MS). For all above ground produce (e.g., apples), peeling did not have a significant effect on the concentration of total arsenic present.

Soil (microbial) disturbance affect the zinc isotope biogeochemistry but has little effect on plant zinc uptake.

Zinc (Zn) is an important micronutrient but can be toxic at elevated concentrations. We conducted an experiment to test the effect of plant growth and soil microbial disturbance on Zn in soil and plants. Pots were prepared with and without maize and in an undisturbed soil, a soil that was disturbed by X-ray sterilization and a soil that was sterilized but reconditioned with the original microbiome. The Zn concentration and isotope fractionation between the soil and the soil pore water increased with time, which is probably due to physical disturbance and fertilization. The presence of maize increased the Zn concentration and isotope fractionation in pore water. This was likely related to the uptake of light isotopes by plants and root exudates that solubilized heavy Zn from the soil. The sterilization disturbance increased the concentration of Zn in the pore water, because of abiotic and biotic changes. Despite a threefold increase in Zn concentration and changes in the Zn isotope composition in the pore water, the Zn content and isotope fractionation in the plant did not change. These results have implications for Zn mobility and uptake in crop plants and are relevant in terms of Zn nutrition.

Plant secondary metabolite-dependent plant-soil feedbacks can improve crop yield in the field.

Plant secondary metabolites that are released into the rhizosphere alter biotic and abiotic soil properties, which in turn affect the performance of other plants. How this type of plant-soil feedback affects agricultural productivity and food quality in the field in the context of crop rotations is unknown. Here, we assessed the performance, yield and food quality of three winter wheat varieties growing in field plots whose soils had been conditioned by either wild type or benzoxazinoid-deficient bx1 maize mutant plants. Following maize cultivation, we detected benzoxazinoid-dependent chemical and microbial fingerprints in the soil. The benzoxazinoid fingerprint was still visible during wheat growth, but the microbial fingerprint was no longer detected. Wheat emergence, tillering, growth, and biomass increased in wild type conditioned soils compared to bx1 mutant conditioned soils. Weed cover was similar between soil conditioning treatments, but insect herbivore abundance decreased in benzoxazinoid-conditioned soils. Wheat yield was increased by over 4% without a reduction in grain quality in benzoxazinoid-conditioned soils. This improvement was directly associated with increased germination and tillering. Taken together, our experiments provide evidence that soil conditioning by plant secondary metabolite producing plants can increase yield via plant-soil feedbacks under agronomically realistic conditions. If this phenomenon holds true across different soils and environments, optimizing root exudation chemistry could be a powerful, genetically tractable strategy to enhance crop yields without additional inputs.

Spatial heterogeneity and kinetic regulation of arsenic dynamics in mangrove sediments: the Sundarbans, Bangladesh.

The biogeochemistry of arsenic (As) in sediments is regulated by multiple factors such as particle size, dissolved organic matter (DOM), iron mobilization, and sediment binding characteristics, among others. Understanding the heterogeneity of factors affecting As deposition and the kinetics of mobilization, both horizontally and vertically, across sediment depositional environments was investigated in Sundarban mangrove ecosystems, Bengal Delta, Bangladesh. Sediment cores were collected from 3 different Sundarbans locations and As concentration down the profiles were found to be more associated with elevated Fe and Mn than with organic matter (OM). At one site chosen for field monitoring, sediment cores, pore and surface water, and in situ diffusive gradients in thin films (DGT) measurements (which were used to model As sediment pore-water concentrations and resupply from the solid phase) were sampled from four different subhabitats. Coarse-textured riverbank sediment porewaters were high in As, but with a limited resupply of As from the solid phase compared to fine-textured and high organic matter content forest floor sediments, where porewater As was low, but with much higher As resupply. Depositional environment (overbank verses forest floor) and biological activity (input of OM from forest biomass) considerably affected As dynamics over very short spatial distances in the mosaic of microhabitats that constitute a mangrove ecosystem.

HPLC-HG-ICP-MS: a sensitive and selective method for inorganic arsenic in seafood.

The addition of an online post-column hydride generation (HG) step to the commonly used high-performance liquid chromatography inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) setup for arsenic speciation proved to significantly improve the detection limits for the determination of inorganic arsenic (iAs) as arsenate in seafood samples, where the limit of detection and limit of quantification were found to be 0.0004 and 0.0014 mg kg(-1), respectively with HG. HG as an additional step further added to the selectivity of the determination of the iAs species and increased the detection and quantification of low levels of iAs (<0.002 mg kg(-1)) in samples with complicated matrices.

Increasing temperature and flooding enhance arsenic release and biotransformations in Swiss soils.

Reductive dissolution is one of the main causes for arsenic (As) mobilisation in flooded soils while biomethylation and biovolatilisation are two microbial mechanisms that greatly influence the mobility and toxicity of As. Climate change results in more extreme weather events such as flooding and higher temperatures, potentially leading to an increase in As release and biotransformations. Here, we investigated the effects of flooding and temperature on As release, biomethylation and biovolatilisation from As-rich soils with different pH and source of As (one acidic and anthropogenic (Salanfe) and one neutral and geogenic (Liesberg)). Flooded soils incubated at 23 °C for two weeks showed a ~ 3-fold (Liesberg site) and ~ 7-fold (Salanfe site) increase in the total As concentration of soil solution compared to those incubated at 18 °C. Methyl- and thio-As species were found in the acidic soil and soil solution. High temperatures enhanced thiolation and methylation although inorganic As was predominant. We also show that volatile As fluxes increased more than 4-fold between treatments, from 18 ± 5 ng/kg/d at 18 °C to 75 ± 6 ng/kg/d at 23 °C from Salanfe soil. Our results suggest that high As soils with acidic pH can become an important source of As to the surrounding environment according to realistic climatic scenarios, and that biovolatilisation is very sensitive to increases in temperature. This study provides new data and further justifies further investigations into climate-induced changes on As release and speciation and its links to important factors such as microbial ecology and sulfate or iron biogeochemistry. SYNOPSIS: In the studied Swiss soils, elevated temperature increases arsenic mobility through volatilisation and methylation.

Organo-mercury species in a polluted agricultural flood plain: Combining speciation methods and polymerase chain reaction to investigate pathways of contamination.

The analysis of organic mercury (Hg) species in polluted soils is a necessary tool to assess the environmental risk(s) of mercury in contaminated legacy sites. The artificial formation of monomethylmercury (MeHg) during soil extraction and/or analysis is a well-known limitation and is especially relevant in highly polluted areas where MeHg/Hg ratios are notoriously low. Although this has been known for almost 30 years, the thorough characterisation of artificial formation rates is rarely a part of the method development in scientific literature. Here we present the application of two separate procedures (inorganic Hg (iHg) spiking and double-spike isotope dilution analyses (DSIDA)) to determine and correct for artificial Hg methylation in MeHg-selective acid-leaching/organic solvent extraction procedure. Subsequently, we combined corrected MeHg and ethylmercury (EtHg) measurements with PCR amplification of hgcA genes to distinguish between naturally formed MeHg from primary deposited MeHg in soils from a legacy site in a Swiss mountain valley. We found the DSIDA procedure incompatible with the organomercury selective extraction method due to the quantitative removal of iHg. Methylation factors from iHg spiking were in the range of (0.0075 ± 0.0001%) and were consistent across soils and sediment matrices. Further, we suggest that MeHg was deposited and not formed in-situ in two out of three studied locations. Our line of evidence consists of 1) the concomitant detection of EtHg, 2) the elevated MeHg concentrations (up to 4.84 µg kg-1), and 3) the absence of hgcA genes at these locations. The combination of Hg speciation and methylation gene (hgcA) abundance analyses are tools suited to assess Hg pollution pathways at Hg legacy sites.

Atmospheric stability of arsine and methylarsines.

Arsenic (As) occurs in a variety of different chemical forms, among them volatile (gaseous) species, usually referred to as arsine and methylarsines. Here we demonstrate that arsine and methylarsines are stable in air in concentrations at the µg/L gas level. We determined half-lives of approximately 8 h under daytime conditions (UV light) for all methylated arsines, while the same species were found to be considerably more stable in night-time (dark) conditions. Arsine (AsH3) showed under both day and night-time conditions, considerably higher stabilities than methylated arsines. We show here that volatile As species seem stable enough to travel considerable distances in the atmosphere from a point source before converting into nonvolatile, oxidized compounds. Also, the degradation pathway leading to the conversion to nonvolatile compounds was investigated using computational chemistry. Arsine and methylarsines' reactions with the hydroxyl radical (•OH) as well as As-C and As-H bonds strengths in the species studied were modeled. Results showed that conversion could not be explained by H abstraction, nor by OH addition. Moreover, it was found that As-C and As-H bonds strengths are not the determining factor responsible for the decrease in stability with ascending methylation of the different volatile arsine species, as previously suggested.

Field fluxes and speciation of arsines emanating from soils.

The biogeochemical cycle of arsenic (As) has been extensively studied over the past decades because As is an environmentally ubiquitous, nonthreshold carcinogen, which is often elevated in drinking water and food. It has been known for over a century that micro-organisms can volatilize inorganic As salts to arsines (arsine AsH(3), mono-, di-, and trimethylarsines, MeAsH(2), Me(2)AsH, and TMAs, respectively), but this part of the As cycle, with the exception of geothermal environs, has been almost entirely neglected because of a lack of suited field measurement approaches. Here, a validated, robust, and low-level field-deployable method employing arsine chemotrapping was used to quantify and qualify arsines emanating from soil surfaces in the field. Up to 240 mg/ha/y arsines was released from low-level polluted paddy soils (11.3 ± 0.9 mg/kg As), primarily as TMAs, whereas arsine flux below method detection limit was measured from a highly contaminated mine spoil (1359 ± 212 mg/kg As), indicating that soil chemistry is vital in understanding this phenomenon. In microcosm studies, we could show that under reducing conditions, induced by organic matter (OM) amendment, a range of soils varied in their properties, from natural upland peats to highly impacted mine-spoils, could all volatilize arsines. Volatilization rates from 0.5 to 70 µg/kg/y were measured, and AsH(3), MeAsH(2), Me(2)AsH, and TMAs were all identified. Addition of methylated oxidated pentavalent As, namely monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA), to soil resulted in elevated yearly rates of volatilization with up to 3.5% of the total As volatilized, suggesting that the initial conversion of inorganic As to MMAA limits the rate of arsine and methylarsines production by soils. The nature of OM amendment altered volatilization quantitatively and qualitatively, and total arsines release from soil showed correlation between the quantity of As and the concentration of dissolved organic carbon (DOC) in the soil porewater. The global flux of arsines emanating from soils was estimated and placed in the context of As atmospheric inputs, with arsines contributing from 0.9 to 2.6% of the global budget.

Validation and deployment of a quantitative trapping method to measure volatile antimony emissions.

Microbial-mediated Sb volatilization is a poorly understood part of the Sb biogeochemical cycle. This is mostly due to a lack of laboratory and field-deployable methods that are capable of quantifying low-level emissions of Sb from diffuse sources. In this study, we validated two methods using a H2O2 -HNO3 liquid chemotrap and an activated coconut shell charcoal solid-phase trap, achieving an absolute limit of detection of 4.6 ng and below 2.0 ng Sb, respectively. The activated charcoal solid-phase trapping method, the most easily operated method, was then applied to contaminated shooting range soils. Four treatments were tested: 1) flooded, 2) manure amended + flooded, 3) 70 % water holding capacity, and 4) manure amendment +70 % water holding capacity, since agricultural practices and flooding events may contribute to Sb volatilization. Volatile Sb was only produced from flooded microcosms and manure amendment greatly influenced the onset and amount of volatile Sb produced. The highest amount of volatile Sb produced, up to 62.1 ng kg-1 d-1, was from the flooded manure amended soil. This suggests that anaerobic microorganisms may potentially be drivers of Sb volatilization. Our results show that polluted shooting range soils are a source of volatile Sb under flooded conditions, which may lead to an increase in the mobility of Sb. Some of these volatile Sb species are toxic and genotoxic, highlighting the role of Sb volatilization on environmental health, especially for individuals living in contaminated areas exposed to wetlands or flooded conditions (e.g., rice paddy agriculture surrounding mining areas). This work paves way for research on Sb volatilization in the environment.

Arsenic dynamics in paddy soil under traditional manuring practices in Bangladesh.

Fertilization with organic matter (farm yard manure and/or rice straw) is thought to enhance arsenic (As) mobilization into soil porewaters, with subsequent As assimilation by rice roots leading to enhanced translocation to the grain. Here, interlinked experiments (field manuring and soil batch culture) were conducted to find the effect of organic matter at a field application rate practiced in Bangladesh (5 t/ha) on As mobilization in soil for paddies impacted by As contaminated groundwater irrigation, a widespread phenomenon in Bangladesh where the experiments were conducted. Total As concentration in a paddy soil (Sonargaon) ranged from 21.9 to 8.1 mg/kg down the soil profile and strongly correlated with TOC content. Arsenic, Fe, Mn, and DOC release into soil solution, and As speciation, are intimately linked to OM amendment, soil depth and temporal variation. Organic matter amendments lead to increased mobilization of As into both soil porewaters and standing surface waters. The As speciation in the porewater was dominated by inorganic As (Asi) (arsenite and arsenate), with traces amounts of methylated species (DMAV and MMAV) only being found with OM amendment. It was noted in field trials that OM fertilization greatly enhanced As mobility to surface waters, which may have major implications for the fate of As in paddy agronomic ecosystems.

250-year records of mercury and trace element deposition in two lakes from Cajas National Park, SW Ecuadorian Andes.

Historical records of trace elements in lake sediments provide source-to-sink information about potentially toxic pollutants across space and time. We investigated two lakes located at different elevations in the Ecuadorian Andes to understand how trace element fluxes are related to (i) geology, (ii) erosion in the watersheds, and (iii) local point sources and atmospheric loads. In remote Lake Fondococha (4150 m a.s.l.), total Hg fluxes stay constant between ca. 1760 and 1950 and show an approximately 4.4-fold increase between pre-1950 and post-1950 values. The post-1950 increase in fluxes of other trace elements (V, Cr, Co, Ni, Cu, Zn, As, Cd, and Pb) is lower (2.1-3.0-fold) than for Hg. Mostly lithogenic sources and enhanced soil erosion contribute to their post-1950 increase (lithogenic contribution: > 85%, Hg: ~ 58%). Average post-1950 Hg fluxes are approximately 4.3 times higher in peri-urban Lake Llaviucu (3150 m a.s.l.) than in the remote Lake Fondococha. Post-1950 fluxes of the other trace elements showed larger differences between Lakes Fondococha and Llaviucu (5.2 < 25-29.5-fold increase; Ni < Pb-Cd). The comparison of the post-1950 average trace element fluxes that are derived from point and airborne sources revealed 5-687 (Hg-Pb) times higher values in Lake Llaviucu than in Lake Fondococha suggesting that Lake Llaviucu's proximity to the city of Cuenca strongly influences its deposition record (industrial emissions, traffic, caged fishery). Both lakes responded with temporary drops in trace element accumulations to park regulations in the 1970s and 1990s, but show again increasing trends in recent times, most likely caused by increase in vehicular traffic and openings of copper and gold mines around Cajas National Park.

A Systematic Analysis of Metal and Metalloid Concentrations in Eight Zebrafish Recirculating Water Systems.

Metals and metalloids are integral to biological processes and play key roles in physiology and metabolism. Nonetheless, overexposure to some metals or lack of others can lead to serious health consequences. In this study, eight zebrafish facilities collaborated to generate a multielement analysis of their centralized recirculating water systems. We report a first set of average concentrations for 46 elements detected in zebrafish facilities. Our results help to establish an initial baseline for trouble-shooting purposes, and in general for safe ranges of metal concentrations in recirculating water systems, supporting reproducible scientific research outcomes with zebrafish.

Emerging investigator series: mercury mobility and methylmercury formation in a contaminated agricultural flood plain: influence of flooding and manure addition.

The fate and the methylation of mercury (Hg) in the terrestrial environment are still poorly understood and although the main drivers of release and methylation of mercury in soils are known (low redox potential and microbial carbon availability) their interactions are not well understood. This is of concern since many agriculturally used floodplains, where the recurring flooding and agricultural practices (e.g. manure amendments) may have an impact on the fate and the biomethylation of Hg, are at the same time Hg-contaminated. In this study, we modified and validated existing methods to extract and analyze methylmercury (MeHg) by HPLC-ICP-MS in soils and we assessed the Hg and MeHg concentrations in three fields situated in a Hg polluted agricultural floodplain. Further, we incubated the top soil from the three studied fields for 11 days under flooded conditions in presence or absence of 2 mass% of cow manure, a common agricultural amendment in the area. Total Hg and MeHg concentrations ranged from