Zinc mediates control of nitrogen fixation via transcription factor filamentation.

Plants adapt to fluctuating environmental conditions by adjusting their metabolism and gene expression to maintain fitness1. In legumes, nitrogen homeostasis is maintained by balancing nitrogen acquired from soil resources with nitrogen fixation by symbiotic bacteria in root nodules2-8. Here we show that zinc, an essential plant micronutrient, acts as an intracellular second messenger that connects environmental changes to transcription factor control of metabolic activity in root nodules. We identify a transcriptional regulator, FIXATION UNDER NITRATE (FUN), which acts as a sensor, with zinc controlling the transition between an inactive filamentous megastructure and an active transcriptional regulator. Lower zinc concentrations in the nodule, which we show occur in response to higher levels of soil nitrate, dissociates the filament and activates FUN. FUN then directly targets multiple pathways to initiate breakdown of the nodule. The zinc-dependent filamentation mechanism thus establishes a concentration readout to adapt nodule function to the environmental nitrogen conditions. In a wider perspective, these results have implications for understanding the roles of metal ions in integration of environmental signals with plant development and optimizing delivery of fixed nitrogen in legume crops.

Effect of a Zinc Phosphate Shell on the Uptake and Translocation of Foliarly Applied ZnO Nanoparticles in Pepper Plants (Capsicum annuum).

Here, isotopically labeled 68ZnO NPs (ZnO NPs) and 68ZnO NPs with a thin 68Zn3(PO4)2 shell (ZnO_Ph NPs) were foliarly applied (40 µg Zn) to pepper plants (Capsicum annuum) to determine the effect of surface chemistry of ZnO NPs on the Zn uptake and systemic translocation to plant organs over 6 weeks. Despite similar dissolution of both Zn-based NPs after 3 weeks, the Zn3(PO4)2 shell on ZnO_Ph NPs (48 ± 12 nm; -18.1 ± 0.6 mV) enabled a leaf uptake of 2.31 ± 0.34 µg of Zn, which is 2.7 times higher than the 0.86 ± 0.18 µg of Zn observed for ZnO NPs (26 ± 8 nm; 14.6 ± 0.4 mV). Further, ZnO_Ph NPs led to higher Zn mobility and phloem loading, while Zn from ZnO NPs was stored in the epidermal tissues, possibly through cell wall immobilization as a storage strategy. These differences led to higher translocation of Zn from the ZnO_Ph NPs within all plant compartments. ZnO_Ph NPs were also more persistent as NPs in the exposed leaf and in the plant stem over time. As a result, the treatment of ZnO_Ph NPs induced significantly higher Zn transport to the fruit than ZnO NPs. As determined by spICP-TOFMS, Zn in the fruit was not in the NP form. These results suggest that the Zn3(PO4)2 shell on ZnO NPs can help promote the transport of Zn to pepper fruits when foliarly applied. This work provides insight into the role of Zn3(PO4)2 on the surface of ZnO NPs in foliar uptake and in planta biodistribution for improving Zn delivery to edible plant parts and ultimately improving the Zn content in food for human consumption.

Biomineralization of mantis shrimp dactyl club following molting: Apatite formation and brominated organic components.

The stomatopod Odontodactylus scyllarus uses weaponized club-like appendages to attack its prey. These clubs are made of apatite, chitin, amorphous calcium carbonate, and amorphous calcium phosphate organized in a highly hierarchical structure with multiple regions and layers. We follow the development of the biomineralized club as a function of time using clubs harvested at specific times since molting. The clubs are investigated using a broad suite of techniques to unravel the biomineralization history of the clubs. Nano focus synchrotron x-ray diffraction and x-ray fluorescence experiments reveal that the club structure is more organized with more sub-regions than previously thought. The recently discovered impact surface has crystallites in a different size and orientation than those in the impact region. The crystal unit cell parameters vary to a large degree across individual samples, which indicates a spatial variation in the degree of chemical substitution. Energy dispersive spectroscopy and Raman spectroscopy show that this variation cannot be explained by carbonation and fluoridation of the lattice alone. X-ray fluorescence and mass spectroscopy show that the impact surface is coated with a thin membrane rich in bromine that forms at very initial stages of club formation. Proteomic studies show that a fraction of the club mineralization protein-1 has brominated tyrosine suggesting that bromination of club proteins at the club surface is an integral component of the club design. Taken together, the data unravel the spatio-temporal changes in biomineral structure during club formation. STATEMENT OF SIGNIFICANCE: Mantis shrimp hunt using club-like appendages that contain apatite, chitin, amorphous calcium carbonate, and amorphous calcium phosphate ordered in a highly hierarchical structure. To understand the formation process of the club we analyze clubs harvested at specific times since molting thereby constructing a club formation map. By combining several methods ranging from position resolved synchrotron X-ray diffraction to proteomics, we reveal that clubs form from an organic membrane with brominated protein and that crystalline apatite phases are present from the very onset of club formation and grow in relative importance over time. This reveals a complex biomineralization process leading to these fascinating biomineralized tools.

Addressing uncertainties in correlative imaging of exogenous particles with the tissue microanatomy with synchronous imaging strategies.

Exposure to exogenous particles is of increasing concern to human health. Characterizing the concentrations, chemical species, distribution, and involvement of the stimulus with the tissue microanatomy is essential in understanding the associated biological response. However, no single imaging technique can interrogate all these features at once, which confounds and limits correlative analyses. Developments of synchronous imaging strategies, allowing multiple features to be identified simultaneously, are essential to assess spatial relationships between these key features with greater confidence. Here, we present data to first highlight complications of correlative analysis between the tissue microanatomy and elemental composition associated with imaging serial tissue sections. This is achieved by assessing both the cellular and elemental distributions in three-dimensional space using optical microscopy on serial sections and confocal X-ray fluorescence spectroscopy on bulk samples, respectively. We propose a new imaging strategy using lanthanide-tagged antibodies with X-ray fluorescence spectroscopy. Using simulations, a series of lanthanide tags were identified as candidate labels for scenarios where tissue sections are imaged. The feasibility and value of the proposed approach are shown where an exposure of Ti was identified concurrently with CD45 positive cells at sub-cellular resolutions. Significant heterogeneity in the distribution of exogenous particles and cells can be present between immediately adjacent serial sections showing a clear need of synchronous imaging methods. The proposed approach enables elemental compositions to be correlated with the tissue microanatomy in a highly multiplexed and non-destructive manner at high spatial resolutions with the opportunity for subsequent guided analysis.

Detection and Characterization of TiO2 Nanomaterials in Sludge from Wastewater Treatment Plants of Chihuahua State, Mexico.

TiO2 nanoparticles (TiO2-NPs) have a wide range of industrial applications (paintings, sunscreens, food and cosmetics) and is one of the most intensively used nanomaterials worldwide. Leaching from commercial products TiO2-NPs are predicted to significantly accumulate in wastewater sludges, which are then often used as soil amendment. In this work, sludge samples from four wastewater treatment plants of the Chihuahua State in Mexico were obtained during spring and summer (2017). A comprehensive characterization study was performed by X-ray based (laboratory and synchrotron) techniques and electron microscopy. Ti was detected in all sludge samples (1810-2760 mg/kg) mainly as TiO2 particles ranging from 40 nm up to hundreds of nm. Micro-XANES data was analyzed by principal component analysis and linear combination fitting enabling the identification of three predominant Ti species: anatase, rutile and ilmenite. Micro-XANES from the smaller Ti particles was predominantly anatase (68% + 32% rutile), suggesting these TiO2-NPs originate from paintings and cosmetics. TEM imaging confirmed the presence of nanoscale Ti with smooth surface morphologies resembling engineered TiO2-NPs. The size and crystalline phase of TiO2-NPs in the sludge from this region suggest increased reactivity and potential toxicity to agro-systems. Further studies should be dedicated to evaluating this.

Assessing implications of nanoplastics exposure to plants with advanced nanometrology techniques.

Despite the increasing attention given to the impacts of nanoplastics in terrestrial environments, there is limited data about the effects on plants, and the quantitative information on uptake. In the present study, wheat plants grown in hydroponics were exposed to Pd-doped nanoplastics. This allowed us to quantify nanoplastics uptake and translocation to the shoots. Visualization of nanoplastics in roots was performed with synchrotron micro X-ray fluorescence (µXRF). Nanoplastics accumulated on the root epidermis, especially at the root tip and in root maturation zones. A close relationship between plant roots, rhizodeposits and nanoplastics behaviour was shown. Reinforcement of the cell wall in roots was evidenced using Fourier transform infrared spectroscopy (FTIR) and synchrotron-computed microtomography (µCT). Synchrotron-computed nanotomography (nanoCT) evidenced the presence of globular structures but they could not be identified as nanoplastics since they were observed both in the control and treated roots. By utilizing the inorganic tracer in the doped-nanoplastics, this study paves the road for elucidating interactions in more complex systems by using an integrative approach combining classical phytotoxicity markers with advanced nanometrology techniques.

Cutting-edge spectroscopy techniques highlight toxicity mechanisms of copper oxide nanoparticles in the aquatic plant Myriophyllum spicatum.

Copper oxide nanoparticles (CuO-NPs) have been increasingly released in aquatic ecosystems over the past decades as they are used in many applications. Cu toxicity to different organisms has already been highlighted in the literature, however toxicity mechanisms of the nanoparticulate form remain unclear. Here, we investigated the effect, transfer and localization of CuO-NPs compared to Cu salt on the aquatic plant Myriophyllum spicatum, an ecotoxicological model species with a pivotal role in freshwater ecosystems, to establish a clear mode of action. Plants were exposed to 0.5 mg/L Cu salt, 5 and 70 mg/L CuO-NPs during 96 h and 10 days. Several morphological and physiological endpoints were measured. Cu salt was found more toxic than CuO-NPs to plants based on all the measured endpoints despite a similar internal Cu concentration demonstrated via Cu mapping by micro particle-induced X-ray emission (µPIXE) coupled to Rutherford backscattering spectroscopy (RBS). Biomacromolecule composition investigated by FTIR converged between 70 mg/L CuO-NPs and Cu salt treatments after 10 days. This demonstrates that the difference of toxicity comes from a sudden massive Cu2+ addition from Cu salt similar to an acute exposure, versus a progressive leaching of Cu2+ from CuO-NPs representing a chronic exposure. Understanding NP toxicity mechanisms can help in the future conception of safer by design NPs and thus diminishing their impact on both the environment and humans.

Correction: Spatiotemporal distribution and speciation of silver nanoparticles in the healing wound.

Correction for 'Spatiotemporal distribution and speciation of silver nanoparticles in the healing wound' by Marco Roman et al., Analyst, 2020, 145, 6456-6469, DOI: 10.1039/D0AN00607F.

Paracyclops chiltoni inhabiting water highly contaminated with arsenic: Water chemistry, population structure, and arsenic distribution within the organism.

We investigated population structure and arsenic bioaccumulation and distribution in zooplankton inhabiting highly contaminated freshwater with arsenic. We collected water and zooplankton samples over a 4 year period, determined environmental temperature as well as water temperature, pH, electrical conductivity (EC), total dissolved solids (TDS), oxidation-reduction potential (ORP), dissolved oxygen (DO), major cations and anions and total arsenic concentration. We identified zooplankton species and determined their abundance, length, sex ratios, and arsenic bioaccumulation and distribution in exposed organisms. At the study site, an extremophile, Paracyclops chiltoni, was found to survive in an environment with high concentration of arsenic, sulfate and fluoride in freshwater as a well-adapted organism. Results showed that the average arsenic concentration in freshwater was 53.64 ± 10.58 mg/L. Exposed organisms of Paracyclops chiltoni showed arsenic accumulation (up to 9.6 ± 5.4 mgAs/kg) in its body, likely in the digestive tract as well as typical abundance and length, which showed a relationship to environmental temperature and oxic conditions in freshwater. Metallotolerant copepods might help to better understand if arsenic methylation processes occur in freshwater aquatic organisms.

Synchrotron X-ray Fluorescence and FTIR Signatures for Amyloid Fibrillary and Nonfibrillary Plaques.

Amyloid plaques are one of the principal hallmarks of Alzheimer's disease and are mainly composed of Aß amyloid peptides together with other components such as lipids, cations, or glycosaminoglycans. The structure of amyloid peptide's aggregates is related to the peptide toxicity and highly depends on the aggregation conditions and the presence of cofactors. While fibrillary aggregates are nowadays considered nontoxic, oligomeric/granular (nonfibrillary) aggregates have been found to be toxic. In this work we have characterized in situ two different types of amyloid deposits analyzing sections of the cortex of patients in advanced stages of Alzheimer disease. By combining SR-µFTIR for the study of the secondary structure of the peptide and ThS fluorescence as an indicator of fibrillary structures, we found two types of plaques: ThS positive plaques with a clear infrared band at 1630 cm-1 that would correspond to fibrillary plaques and ThS negative plaques showing a mixture of nonfibrillar ß-sheet and unordered aggregated structures that would correspond to the nonfibrillary plaques (plaques with increased unordered structure). The analysis of the FTIR spectra has allowed correlation of lipid oxidation with the presence of nonfibrillary plaques. The metal composition of the two types of plaques has been analyzed using SR-nano-XRF and XANES. The results have shown higher accumulation of iron (mainly Fe2+) in fibrillary plaques than in nonfibrillary ones. However, in nonfibrillary plaques Fe3+ has been found to predominate over Fe2+. The identification of different types of aggregated forms and the different composition of metals found in the different types of plaques could be of paramount importance for the understanding of the development of Alzheimer disease.

Synchrotron Radiation Spectroscopy and Transmission Electron Microscopy Techniques to Evaluate TiO2 NPs Incorporation, Speciation, and Impact on Root Cells Ultrastructure of Pisum sativum L. Plants.

Biosolids (Bs) for use in agriculture are an important way for introducing and transferring TiO2 nanoparticles (NPs) to plants and food chain. Roots of Pisum sativum L. plants grown in Bs-amended soils spiked with TiO2 800 mg/kg as rutile NPs, anatase NPs, mixture of both NPs and submicron particles (SMPs) were investigated by Transmission Electron Microscopy (TEM), synchrotron radiation based micro X-ray Fluorescence and micro X-ray Absorption Near-Edge Structure (µXRF/µXANES) and Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). TEM analysis showed damages in cells ultrastructure of all treated samples, although a more evident effect was observed with single anatase or rutile NPs treatments. Micro-XRF and TEM evidenced the presence of nano and SMPs mainly in the cortex cells near the rhizodermis. Micro-XRF/micro-XANES analysis revealed anatase, rutile, and ilmenite as the main TiO2 polymorphs in the original soil and Bs, and the preferential anatase uptake by the roots. For all treatments Ti concentration in the roots increased by 38-56%, however plants translocation factor (TF) increased mostly with NPs treatment (261-315%) and less with SMPs (about 85%), with respect to control. In addition, all samples showed a limited transfer of TiO2 to the shoots (very low TF value). These findings evidenced a potential toxicity of TiO2 NPs present in Bs and accumulating in soil, suggesting the necessity of appropriate regulations for the occurrence of NPs in Bs used in agriculture.

A Critical Review on the Impacts of Nanoplastics and Microplastics on Aquatic and Terrestrial Photosynthetic Organisms.

Microplastic and nanoplastic contamination is widespread and affects aquatic and terrestrial ecosystems. Photosynthetic organisms are present in both media, they are primary producers, sink for CO2 , and they represent a major point of entry in the food chain. Here, the current knowledge on the fate and impacts of microplastics and nanoplastics in interaction with these organisms is reviewed. As a general trend, plastic characteristics (smaller size and positive charge) play a crucial role in their toxicity toward photosynthetic organisms. Plastic leachates (containing additives) also represent a major source of toxicity, and some harmful compounds such as phthalate esters are shown to accumulate in plants and generate a risk for the consumers. Adsorption of plastic particles is evidenced for each type of photosynthetic organism, and uptake and translocation in terrestrial plants is evidenced for nanoplastics, leading to concerns for trophic chain contamination. The available techniques for the detection of microplastics and nanoplastics and their secondary products in biological samples and media are also listed. Finally, the current gaps of knowledge, specific challenges, and future research directions are also discussed.

Spatiotemporal distribution and speciation of silver nanoparticles in the healing wound.

The medical application of nanomaterials is growing fast. Amongst the most widely used, silver nanoparticles are antimicrobial agents whose key application is the care of burns and chronic wounds. Still, their absorption, distribution, metabolism and excretion behaviour in vivo has not yet been systematically investigated. We collected full-profile specimens of skin from four hospital patients with mid-to-deep thickness burns or equivalent skin wounds, treated with dressings containing silver nanoparticles or silver sulfadiazine. Synchrotron radiation µXRF/µXANES and laser ablation-ICP-MS were used to provide the first semi-quantitative/high resolution direct information on the spatiotemporal distribution and speciation of silver in vivo. The metal was rapidly released onto the wound surface, followed by a significant structure-dependent penetration into the damaged tissues. This was accompanied by sequential processes of metallic silver dissolution, chloride complexation, change to metal-thiol protein complexes, and final mobilization into deeper skin layers towards the vascular networks. Complete local clearance of silver was observed after 12 days of treatment in the case of full healing. The results provide a complete insight into the dynamics of silver in real human wounds, and a new basis for the design of innovative silver nanomaterials with optimal antibacterial efficacy and minimized risk for the patient.

Effect of phosphorus supply on root traits of two Brassica oleracea L. genotypes.

BACKGROUND: Phosphorus (P) deficiency limits crop production worldwide. Crops differ in their ability to acquire and utilise the P available. The aim of this study was to determine root traits (root exudates, root system architecture (RSA), tissue-specific allocation of P, and gene expression in roots) that (a) play a role in P-use efficiency and (b) contribute to large shoot zinc (Zn) concentration in Brassica oleracea. RESULTS: Two B. oleracea accessions (var. sabellica C6, a kale, and var. italica F103, a broccoli) were grown in a hydroponic system or in a high-throughput-root phenotyping (HTRP) system where they received Low P (0.025 mM) or High P (0.25 mM) supply for 2 weeks. In hydroponics, root and shoot P and Zn concentrations were measured, root exudates were profiled using both Fourier-Transform-Infrared spectroscopy and gas-chromatography-mass spectrometry and previously published RNAseq data from roots was re-examined. In HTRP experiments, RSA (main and lateral root number and lateral root length) was assessed and the tissue-specific distribution of P was determined using micro-particle-induced-X-ray emission. The C6 accession had greater root and shoot biomass than the F103 accession, but the latter had a larger shoot P concentration than the C6 accession, regardless of the P supply in the hydroponic system. The F103 accession had a larger shoot Zn concentration than the C6 accession in the High P treatment. Although the F103 accession had a larger number of lateral roots, which were also longer than in the C6 accession, the C6 accession released a larger quantity and number of polar compounds than the F103 accession. A larger number of P-responsive genes were found in the Low P treatment in roots of the F103 accession than in roots of the C6 accession. Expression of genes linked with "phosphate starvation" was up-regulated, while those linked with iron homeostasis were down-regulated in the Low P treatment. CONCLUSIONS: The results illustrate large within-species variability in root acclimatory responses to P supply in the composition of root exudates, RSA and gene expression, but not in P distribution in root cross sections, enabling P sufficiency in the two B. oleracea accessions studied.

Medicago truncatula Ferroportin2 mediates iron import into nodule symbiosomes.

Iron is an essential cofactor for symbiotic nitrogen fixation, required by many of the enzymes involved, including signal transduction proteins, O2 homeostasis systems, and nitrogenase itself. Consequently, host plants have developed a transport network to deliver essential iron to nitrogen-fixing nodule cells. Ferroportin family members in model legume Medicago truncatula were identified and their expression was determined. Yeast complementation assays, immunolocalization, characterization of a tnt1 insertional mutant line, and synchrotron-based X-ray fluorescence assays were carried out in the nodule-specific M. truncatula ferroportin Medicago truncatula nodule-specific gene Ferroportin2 (MtFPN2) is an iron-efflux protein. MtFPN2 is located in intracellular membranes in the nodule vasculature and in inner nodule tissues, as well as in the symbiosome membranes in the interzone and early-fixation zone of the nodules. Loss-of-function of MtFPN2 alters iron distribution and speciation in nodules, reducing nitrogenase activity and biomass production. Using promoters with different tissular activity to drive MtFPN2 expression in MtFPN2 mutants, we determined that expression in the inner nodule tissues is sufficient to restore the phenotype, while confining MtFPN2 expression to the vasculature did not improve the mutant phenotype. These data indicate that MtFPN2 plays a primary role in iron delivery to nitrogen-fixing bacteroids in M. truncatula nodules.

In Vivo Bioengineering of Fluorescent Conductive Protein-Dye Microfibers.

Engineering protein-based biomaterials is extremely challenging in bioelectronics, medicine, and materials science, as mechanical, electrical, and optical properties need to be merged to biocompatibility and resistance to biodegradation. An effective strategy is the engineering of physiological processes in situ, by addition of new properties to endogenous components. Here we show that a green fluorescent semiconducting thiophene dye, DTTO, promotes, in vivo, the biogenesis of fluorescent conductive protein microfibers via metabolic pathways. By challenging the simple freshwater polyp Hydra vulgaris with DTTO, we demonstrate the stable incorporation of the dye into supramolecular protein-dye co-assembled microfibers without signs of toxicity. An integrated multilevel analysis including morphological, optical, spectroscopical, and electrical characterization shows electrical conductivity of biofibers, opening the door to new opportunities for augmenting electronic functionalities within living tissue, which may be exploited for the regulation of cell and animal physiology, or in pathological contexts to enhance bioelectrical signaling.

Effects of a nitrification inhibitor on nitrogen species in the soil and the yield and phosphorus uptake of maize.

Phosphorus (P) resource availability is declining and the efficiency of applied nutrients in agricultural soils is becoming increasingly important. This is especially true for P fertilizers from recycled materials, which often have low plant availability. Specific co-fertilization with ammonium can enhance P plant availability in soils amended with these P fertilizers, and thus the yield of plants. To investigate this effect, we performed a pot experiment with maize in slightly acidic soil (pH 6.9) with one water-soluble (triple superphosphate [TSP]) and two water-insoluble (sewage sludge-based and hyperphosphate [Hyp]) P fertilizers and an ammonium sulfate nitrate with or without a nitrification inhibitor (NI). The dry matter yield of maize was significantly increased by the NI with the Hyp (from 14.7 to 21.5 g/pot) and TSP (from 40.0 to 45.4 g/pot) treatments. Furthermore, P uptake was slightly increased in all three P treatments with the NI, but not significantly. Olsen-P extraction and P K-edge micro-X-ray absorption near-edge structure (XANES) spectroscopy showed that apatite-P of the water-insoluble P fertilizers mobilized during the plant growth period. In addition, novel nitrogen (N) K-edge micro-XANES spectroscopy and the Mogilevkina method showed that the application of an NI increased the fixation of ammonium in detectable hot spots in the soil. Thus, the delay in the nitrification process by the NI and the possible slow-release of temporarily fixed ammonium in the soil resulted in a high amount of plant available ammonium in the soil solution. This development probably decreases the rhizosphere pH due to release of H+ by plants during ammonium uptake, which mobilizes phosphorus in the amended soil and increases the dry matter yield of maize. This is especially important for water-insoluble apatite-based P fertilizers (conventional and recycled), which tend to have poor plant availability.

Mineral Element Composition in Grain of Awned and Awnletted Wheat (Triticum aestivum L.) Cultivars: Tissue-Specific Iron Speciation and Phytate and Non-Phytate Ligand Ratio.

In wheat (Triticum aestivum L.), the awns-the bristle-like structures extending from lemmas-are photosynthetically active. Compared to awned cultivars, awnletted cultivars produce more grains per unit area and per spike, resulting in significant reduction in grain size, but their mineral element composition remains unstudied. Nine awned and 11 awnletted cultivars were grown simultaneously in the field. With no difference in 1000-grain weight, a larger calcium and manganese-but smaller iron (Fe) concentrations-were found in whole grain of awned than in awnletted cultivars. Micro X-ray absorption near edge structure analysis of different tissues of frozen-hydrated grain cross-sections revealed that differences in total Fe concentration were not accompanied by differences in Fe speciation (64% of Fe existed as ferric and 36% as ferrous species) or Fe ligands (53% were phytate and 47% were non-phytate ligands). In contrast, there was a distinct tissue-specificity with pericarp containing the largest proportion (86%) of ferric species and nucellar projection (49%) the smallest. Phytate ligand was predominant in aleurone, scutellum and embryo (72%, 70%, and 56%, respectively), while nucellar projection and pericarp contained only non-phytate ligands. Assuming Fe bioavailability depends on Fe ligands, we conclude that Fe bioavailability from wheat grain is tissue specific.

Cadmium distribution in mature durum wheat grains using dissection, laser ablation-ICP-MS and synchrotron techniques.

Understanding how essential and toxic elements are distributed in cereal grains is a key to improving the nutritional quality of cereal-based products. The main objective of this work was to characterize the distribution of Cd and of nutrients (notably Cu, Fe, Mn, P, S and Zn) in the durum wheat grain. Laser ablation inductively coupled mass spectrometry and synchrotron micro X-ray fluorescence were used for micro-scale mapping of Cd and nutrients. A dissection approach was used to quantitatively assess the distribution of Cd and nutrients among grain tissues. Micro X-ray absorption near-edge spectroscopy was used to identify the Cd chemical environment in the crease. Cadmium distribution was characterized by strong accumulation in the crease and by non-negligible dissemination in the endosperm. Inside the crease, Cd accumulated most in the pigment strand where it was mainly associated with sulfur ligands. High-resolution maps highlighted very specific accumulation areas of some nutrients in the germ, for instance Mo in the root cortex primordia and Cu in the scutellum. Cadmium loading into the grain appears to be highly restricted. In the grain, Cd co-localized with several nutrients, notably Mn and Zn, which challenges the idea of selectively removing Cd-enriched fractions by dedicated milling process.

Pyrenoidal sequestration of cadmium impairs carbon dioxide fixation in a microalga.

Mixotrophic microorganisms are able to use organic carbon as well as inorganic carbon sources and thus, play an essential role in the biogeochemical carbon cycle. In aquatic ecosystems, the alteration of carbon dioxide (CO2 ) fixation by toxic metals such as cadmium - classified as a priority pollutant - could contribute to the unbalance of the carbon cycle. In consequence, the investigation of cadmium impact on carbon assimilation in mixotrophic microorganisms is of high interest. We exposed the mixotrophic microalga Chlamydomonas reinhardtii to cadmium in a growth medium containing both CO2 and labelled 13 C-[1,2] acetate as carbon sources. We showed that the accumulation of cadmium in the pyrenoid, where it was predominantly bound to sulphur ligands, impaired CO2 fixation to the benefit of acetate assimilation. Transmission electron microscopy (TEM)/X-ray energy dispersive spectroscopy (X-EDS) and micro X-ray fluorescence (µXRF)/micro X-ray absorption near-edge structure (µXANES) at Cd LIII- edge indicated the localization and the speciation of cadmium in the cellular structure. In addition, nanoscale secondary ion mass spectrometry (NanoSIMS) analysis of the 13 C/12 C ratio in pyrenoid and starch granules revealed the origin of carbon sources. The fraction of carbon in starch originating from CO2 decreased from 73 to 39% during cadmium stress. For the first time, the complementary use of high-resolution elemental and isotopic imaging techniques allowed relating the impact of cadmium at the subcellular level with carbon assimilation in a mixotrophic microalga.