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.

Perinatal foodborne titanium dioxide exposure-mediated dysbiosis predisposes mice to develop colitis through life.

BACKGROUND: Perinatal exposure to titanium dioxide (TiO2), as a foodborne particle, may influence the intestinal barrier function and the susceptibility to develop inflammatory bowel disease (IBD) later in life. Here, we investigate the impact of perinatal foodborne TiO2 exposure on the intestinal mucosal function and the susceptibility to develop IBD-associated colitis. Pregnant and lactating mother mice were exposed to TiO2 until pups weaning and the gut microbiota and intestinal barrier function of their offspring was assessed at day 30 post-birth (weaning) and at adult age (50 days). Epigenetic marks was studied by DNA methylation profile measuring the level of 5-methyl-2'-deoxycytosine (5-Me-dC) in DNA from colic epithelial cells. The susceptibility to develop IBD has been monitored using dextran-sulfate sodium (DSS)-induced colitis model. Germ-free mice were used to define whether microbial transfer influence the mucosal homeostasis and subsequent exacerbation of DSS-induced colitis. RESULTS: In pregnant and lactating mice, foodborne TiO2 was able to translocate across the host barriers including gut, placenta and mammary gland to reach embryos and pups, respectively. This passage modified the chemical element composition of foetus, and spleen and liver of mothers and their offspring. We showed that perinatal exposure to TiO2 early in life alters the gut microbiota composition, increases the intestinal epithelial permeability and enhances the colonic cytokines and myosin light chain kinase expression. Moreover, perinatal exposure to TiO2 also modifies the abilities of intestinal stem cells to survive, grow and generate a functional epithelium. Maternal TiO2 exposure increases the susceptibility of offspring mice to develop severe DSS-induced colitis later in life. Finally, transfer of TiO2-induced microbiota dysbiosis to pregnant germ-free mice affects the homeostasis of the intestinal mucosal barrier early in life and confers an increased susceptibility to develop colitis in adult offspring. CONCLUSIONS: Our findings indicate that foodborne TiO2 consumption during the perinatal period has negative long-lasting consequences on the development of the intestinal mucosal barrier toward higher colitis susceptibility. This demonstrates to which extent environmental factors influence the microbial-host interplay and impact the long-term mucosal homeostasis.

Transcriptional response of Cu-deficient barley (Hordeum vulgare L.) to foliar-applied nano-Cu: Molecular crosstalk between Cu loading into plants and changes in Cu homeostasis genes.

For safe and effective nutrient management, the cutting-edge approaches to plant fertilization are continuously developed. The aim of the study was to analyze the transcriptional response of barley suffering from Cu deficiency to foliar application of nanoparticulate Cu (nano-Cu) and its ionic form (CuSO4) at 100 and 1000 mg L-1 for the examination of their supplementing effect. The initial interactions of Cu-compounds with barley leaves were analyzed with spectroscopic (ICP-OES) and microscopic (SEM-EDS) methods. To determine Cu cellular status, the impact of Cu-compounds on the expression of genes involved in regulating Cu homeostasis (PAA1, PAA2, RAN1, COPT5), aquaporins (NIP2.1, PIP1.1, TIP1.1, TIP1.2) and antioxidant defense response (SOD CuZn, SOD Fe, SOD Mn, CAT) after 1 and 7 days of exposure was analyzed. Although Cu accumulation in plant leaves was detected overtime, the Cu content in leaves exposed to nano-Cu for 7 days was 44.5% lower than in CuSO4 at 100 mg L-1. However, nano-Cu aggregates remaining on the leaf surface indicated a potential difference between measured Cu content and the real Cu pool present in the plant. Our study revealed significant changes in the pattern of gene expression overtime depending on Cu-compound type and dose. Despite the initial puzzling patterns of gene expression, after 7 days all Cu transporters showed significant down-regulation under Cu-compounds exposure to prevent Cu excess in plant cells. Conversely, aquaporin gene expression was induced after 7 days, especially by nano-Cu and CuSO4 at 100 mg L-1 due to the stimulatory effect of low Cu doses. Our study revealed that the gradual release of Cu ions from nano-Cu at a lower rate provided a milder molecular response than CuSO4. It might indicate that nano-Cu maintained better metal balance in plants than the conventional compounds, thus may be considered as a long-term supplier of Cu.

Impact of potentially toxic elements on pines in a former ore processing mine: Exploitation of hyperspectral response from needle and canopy scales.

Anthropic potentially toxic element (PTE) releases can lead to persistent pollution in soil. Monitoring PTEs by their detection and quantification on large scale is of great interest. The vegetation exposed to PTEs can exhibit a reduction of physiological activities, structural damage … Such vegetation trait changes impact the spectral signature in the reflective domain 0.4-2.5 µm. The objective of this study is to characterize the impact of PTEs on the spectral signature of two pine species (Aleppo and Stone pines) in the reflective domain and ensure their assessment. The study focuses on nine PTEs: As, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Zn. The spectra are measured by an in-field spectrometer and an aerial hyperspectral instrument on a former ore processing site. They are completed by measurements related to vegetation traits at needle and tree scales (photosynthetic pigments, dry matter, morphometry …) to define the most sensitive vegetation parameter to each PTE in soil. A result of this study is that chlorophylls and carotenoids are the most correlated to PTE contents. Context-specific spectral indices are specified and used to assess metal contents in soil by regression. These new vegetation indices are compared at needle and canopy scales to literature indices. Most of the PTE contents are predicted at both scales with Pearson correlation scores between 0.6 and 0.9, depending on species and scale.

Interactive effects of metals and carbon nanotubes in a microcosm agrosystem.

Agricultural soils are exposed to multiple contaminants through the use of agrochemicals or sewage sludge, introducing metals, nanomaterials and others. Among nanomaterials, carbon nanotubes (CNTs) are known for their large surface area and adsorption capabilities, possibly modifying other element behavior. However, to date, very little is known about the impacts of such interactions in agrosystems. In this study, we aimed at understanding the transfer and toxicity of contaminants (Cd, Pb, Zn and CNTs) in microcosms including native soil bacteria, earthworms and lettuce. After a 6 week exposure, no effect of the addition of CNTs to metal contaminated soils was detected on bacterial concentration or earthworm growth. However, in lettuce, an interactive effect between CNTs and metals was highlighted: in the soil containing the highest metal concentrations the addition of 0.1 mg kg-1 CNTs led to a biomass loss (-22%) and a flavonoid concentration increase (+27%). In parallel, the addition of CNTs led to differential impacts on elemental uptake in lettuce leaves possibly related to the soil organic matter content. For earthworms, the addition of 10 mg kg-1 CNTs resulted in an increased body elemental transfer in the soil with the higher organic matter content (Pb: + 34% and Zn: + 25%).

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.

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.

Comparative study of response of four crop species exposed to carbon nanotube contamination in soil.

Crop plants are exposed to a variety of contaminants through sewage sludge spreading but very little is known about the impact of emerging contaminants such as nanomaterials. To date their impact on plants is still very controversial with many works claiming negative impacts while some authors suggest their use as plant growth regulator in agriculture. In this study, aiming to better understand where these discrepancies may come from, we investigated the influence of plant species (tomato, rapeseed, cucumber and maize) on plant response to a carbon nanotube contamination in soil condition. Our results demonstrate that the same CNT contamination can lead to different effects depending on plant species with positive impacts on cucumber and rapeseed (more than 50% increase in leaf biomass and surface area and 29% increase in chlorophyll for cucumber) but negative impact on maize (-14% for plant height), while tomato was insensitive. FTIR analysis of biomacromolecule composition suggested that these differences could be related with plant cell wall composition (in particular: pectins, xyloglucans and lignins). As a summary, no overall conclusion can be drawn about the toxicity of a specific nanomaterial for all plant species.

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.

Genotypes of the aquatic plant Myriophyllum spicatum with different growth strategies show contrasting sensitivities to copper contamination.

Genotypic variability has been considered for years as a key attribute in species adaptation to new environments. It has been extensively studied in a context of chemical resistance, but remains poorly studied in response to chemical exposure in a context of global change. As aquatic ecosystems are particularly affected by environmental changes, we aimed to study how genotypic variability could inflect the sensitivity of aquatic plants to chemicals. Seven genotypes of Myriophyllum spicatum were exposed to three copper concentrations at 0, 0.15 and 0.5 mg/L. The sensitivity of the different genotypes was assessed through several endpoints such as relative growth rate (RGR) and morphological traits, as well as physiological markers, such as plant biomacromolecular composition. Our results showed that genotypes exhibited significant differences in their life-history traits in absence of chemical contamination. Some trait syndromes were observed, and three growth strategies were identified: (1) biomass production and main shoot elongation, (2) dry matter storage with denser whorls to promote resource conservation and (3) lateral shoot production. An up to eightfold difference in sensitivity for growth-related endpoints was observed among genotypes. Differences in sensitivity were partly attributed to morphological life-history traits. Our results confirm that genotypic variability can significantly affect M. spicatum sensitivity to Cu, and may influence the outcomes of laboratory testing based on the study of one single genotype. We recommend including genotypic variation as an assessment factor in ecological risk assessment and to study this source of variability more in depth as a possible driver of ecosystem resilience.

Does intraspecific variability matter in ecological risk assessment? Investigation of genotypic variations in three macrophyte species exposed to copper.

To limit anthropogenic impact on ecosystems, regulations have been implemented along with global awareness that human activities are harmful to the environment. Ecological risk assessment (ERA) is the main procedure which allows to assess potential impacts of stressors on the environment as a result of human activities. ERA is typically implemented through different steps of laboratory testing. The approaches taken for ERA evolve along with scientific knowledge, to improve predictions on ecological risks for ecosystems. We here address the importance of intraspecific variability as a potential source of error in the laboratory evaluation of pollutants. To answer this question, three aquatic macrophyte species with different life-history traits but with their leaves directly in contact with the water were chosen; Lemna minor and Myriophyllum spicatum, two OECD model species, and Ceratophyllum demersum. For each species, three or four genotypes were exposed to 7-8 copper concentrations (up to 1.9 mg/L, 2 mg/L or 36 mg/L for C. demersum, L. minor and M. spicatum, respectively). To assess species sensitivity, growth-related endpoints such as Relative Growth Rate (RGR), based either on biomass production or on length/frond production, and chlorophyll fluorescence Fv/Fm, were measured. For each endpoint, the effective concentration 50% (EC50) was calculated. Almost all endpoints were affected by Cu exposure, except Fv/Fm of M. spicatum, and resulted in significant differences among genotypes for Cu sensitivity. Genotypes of L. minor exhibited up to 35% of variation in EC50 values based on Fv/Fm, showing differential sensivity among genotypes. Significant differences in EC50 values were found for RGR based on length for M. spicatum, with up to 72% of variation. Finally, C. demersum demonstrated significant sensitivity differences among genotypes with up to 78% variation for EC50 based on length. Overall, interspecific variation was higher than intraspecific variation, and explained 77% of the variation found among genotypes for RGR based on biomass, and 99% of the variation found for Fv/Fm. Our results highlight that depending on the endpoint, sensitivity can vary greatly within a species, and that pollutant- and species-specific endpoints should be considered in ERA.

Environmental variations mediate duckweed (Lemna minor L.) sensitivity to copper exposure through phenotypic plasticity.

Environmentally mediated sensitivity of Lemna minor to copper (Cu) was evaluated for the first time in three experiments: the effects of two levels of nutrient concentration, light irradiance or Cu pre-exposure were tested. Various Cu concentrations (ranging from 0.05 to 0.25 mg/L) were used to assess the sensitivity of L. minor to this metal, using one common strain previously acclimatized to two different levels of light intensity, nutrient enrichment and Cu pre-exposure. Our results showed a phenotypic plastic response of the relative growth rates based on frond number and fresh mass production, and maximum quantum yield of photosystem II (Fv/Fm). Growth was affected by the three environmental conditions both prior and during Cu exposure, whereas Fv/Fm was mostly affected during Cu exposure. Copper significantly influenced all the parameters measured in the three experiments. Environmental conditions significantly modified L. minor sensitivity to Cu in all experiments, with up to twofold difference depending on the treatment. Growth rate was the parameter that was most impacted. Our study revealed for the first time the existence of phenotypic plasticity in L. minor sensitivity to chemical contamination, and implies that environmental context needs to be taken into account for a relevant risk assessment.

Assessing the impacts of sewage sludge amendment containing nano-TiO2 on tomato plants: A life cycle study.

Increasing evidence indicates the presence of engineered nanoparticles (ENPs) in sewage sludge derived from wastewater treatment. Land application of sewage sludge is, therefore, considered as an important pathway for ENP transfer to the environment. The aim of this work was to understand the effects of sewage sludge containing nano-TiO2 on plants (tomato) when used as an amendment in agricultural soil. We assessed developmental parameters for the entire plant life cycle along with metabolic and bio-macromolecule changes and titanium accumulation in plants. The results suggest that the sewage sludge amendment containing nano-TiO2 increased plant growth (142% leaf biomass, 102% fruit yield), without causing changes in biochemical responses, except for a 43% decrease in leaf tannin concentration. Changes in elemental concentrations (mainly Fe, B, P, Na, and Mn) of plant stem, leaves and, to a lesser extent fruits were observed. Fourier-transformed infrared analysis showed maximum changes in plant leaves (decrease in tannins and lignins and increase in carbohydrates) but no change in fruits. No significant Ti enrichment was detected in tomato fruits. In conclusion, we evidenced no acute toxicity to plants and no major implication for food safety after one plant life cycle exposure.

Nicotianamine Synthase 2 Is Required for Symbiotic Nitrogen Fixation in Medicago truncatula Nodules.

Symbiotic nitrogen fixation carried out by the interaction between legumes and diazotrophic bacteria known as rhizobia requires relatively large levels of transition metals. These elements are cofactors of many key enzymes involved in this process. Metallic micronutrients are obtained from soil by the roots and directed to sink organs by the vasculature, in a process mediated by a number of metal transporters and small organic molecules that facilitate metal delivery in the plant fluids. Among the later, nicotianamine is one of the most important. Synthesized by nicotianamine synthases (NAS), this molecule forms metal complexes participating in intracellular metal homeostasis and long-distance metal trafficking. Here we characterized the NAS2 gene from model legume Medicago truncatula. MtNAS2 is located in the root vasculature and in all nodule tissues in the infection and fixation zones. Symbiotic nitrogen fixation requires of MtNAS2 function, as indicated by the loss of nitrogenase activity in the insertional mutant nas2-1, phenotype reverted by reintroduction of a wild-type copy of MtNAS2. This would result from the altered iron distribution in nas2-1 nodules shown with X-ray fluorescence. Moreover, iron speciation is also affected in these nodules. These data suggest a role of nicotianamine in iron delivery for symbiotic nitrogen fixation.

Transfer and Ecotoxicity of Titanium Dioxide Nanoparticles in Terrestrial and Aquatic Ecosystems: A Microcosm Study.

With the advancement in nanotechnology, particularly the use of TiO2 nanoparticles (NPs), there is a need to study their release into the environment and assess the related risk in an environmentally relevant contamination scenario. In the present study, the transfer and toxicity of TiO2 NPs in microcosms mimicking terrestrial and aquatic ecosystems were evaluated. The contaminated soil was prepared by spiking natural soils, with these then used as the basis for all exposure systems including preparation of soil leachates for amphibian exposure. Results demonstrated significant reductions in bacterial (-45%) and archaeal (-36%) nitrifier abundance; significant translocation of Ti to M. truncatula leaves (+422%); significant reductions in plant height (-17%), number of leaves (-29%), and aboveground biomass (-53%); nonsignificant Ti uptake in snail foot and viscera, and excretion in feces; and genotoxicity to X. laevis larvae (+119% micronuclei). Our study highlights a possible risk of engineered TiO2 NPs in the environment in terms of trophic transfer and toxicity in both terrestrial and aquatic environments.

Etiolated Seedling Development Requires Repression of Photomorphogenesis by a Small Cell-Wall-Derived Dark Signal.

Etiolated growth in darkness or the irreversible transition to photomorphogenesis in the light engages alternative developmental programs operating across all organs of a plant seedling. Dark-grown Arabidopsis de-etiolated by zinc (dez) mutants exhibit morphological, cellular, metabolic, and transcriptional characteristics of light-grown seedlings. We identify the causal mutation in TRICHOME BIREFRINGENCE encoding a putative acyl transferase. Pectin acetylation is decreased in dez, as previously found in the reduced wall acetylation2-3 mutant, shown here to phenocopy dez. Moreover, pectin of dez is excessively methylesterified. The addition of very short fragments of homogalacturonan, tri-galacturonate, and tetra-galacturonate, restores skotomorphogenesis in dark-grown dez and similar mutants, suggesting that the mutants are unable to generate these de-methylesterified pectin fragments. In combination with genetic data, we propose a model of spatiotemporally separated photoreceptive and signal-responsive cell types, which contain overlapping subsets of the regulatory network of light-dependent seedling development and communicate via a pectin-derived dark signal.

Medicago truncatula Zinc-Iron Permease6 provides zinc to rhizobia-infected nodule cells.

Zinc is a micronutrient required for symbiotic nitrogen fixation. It has been proposed that in model legume Medicago truncatula, zinc is delivered by the root vasculature into the nodule and released in the infection/differentiation zone. There, transporters must introduce this element into rhizobia-infected cells to metallate the apoproteins that use zinc as a cofactor. MtZIP6 (Medtr4g083570) is an M. truncatula Zinc-Iron Permease (ZIP) that is expressed only in roots and nodules, with the highest expression levels in the infection/differentiation zone. Immunolocalization studies indicate that it is located in the plasma membrane of nodule rhizobia-infected cells. Down-regulating MtZIP6 expression levels with RNAi does not result in any strong phenotype when plants are fed mineral nitrogen. However, these plants displayed severe growth defects when they depended on nitrogen fixed by their nodules, losing of 80% of their nitrogenase activity. The reduction of this activity was likely an indirect effect of zinc being retained in the infection/differentiation zone and not reaching the cytosol of rhizobia-infected cells. These data are consistent with a model in which MtZIP6 would be responsible for zinc uptake by rhizobia-infected nodule cells in the infection/differentiation zone.

Practical review on the use of synchrotron based micro- and nano- X-ray fluorescence mapping and X-ray absorption spectroscopy to investigate the interactions between plants and engineered nanomaterials.

The increased use of engineered nanomaterials (ENMs) in commercial products and the continuous development of novel applications, is leading to increased intentional and unintentional release of ENMs into the environment with potential negative impacts. Particularly, the partition of nanoparticles (NPs) to waste water treatment plant (WWTP) sludge represents a potential threat to agricultural ecosystems where these biosolids are being applied as fertilizers. Moreover, several applications of ENMs in agriculture and soil remediation are suggested. Therefore, detailed risk assessment should be done to evaluate possible secondary negative impacts. The impact of ENMS on plants as central component of ecosystems and worldwide food supply is of primary relevance. Understanding the fate and physical and chemical modifications of NPs in plants and their possible transfer into food chains requires specialized analytical techniques. Due to the importance of both chemical and physical factors to consider for a better understanding of ENMs behavior in complex matrices, these materials can be considered a new type of analyte. An ideal technique should require minimal sample preparation, be non-destructive, and offer the best balance between sensitivity, chemical specificity, and spatial resolution. Synchrotron radiation (SR) techniques are particularly adapted to investigate localization and speciation of ENMs in plants. SR X-ray fluorescence mapping (SR-XFM) offers multi-elemental detection with lateral resolution down to the tens of nm, in combination with spatially resolved X-ray absorption spectroscopy (XAS) speciation. This review will focus on important methodological aspects regarding sample preparation, data acquisition and data analysis of SR-XFM/XAS to investigate interactions between plants and ENMs.

Mother-plant-mediated pumping of zinc into the developing seed.

Insufficient intake of zinc and iron from a cereal-based diet is one of the causes of 'hidden hunger' (micronutrient deficiency), which affects some two billion people(1,2). Identifying a limiting factor in the molecular mechanism of zinc loading into seeds is an important step towards determining the genetic basis for variation of grain micronutrient content and developing breeding strategies to improve this trait(3). Nutrients are translocated to developing seeds at a rate that is regulated by transport processes in source leaves, in the phloem vascular pathway, and at seed sinks. Nutrients are released from a symplasmic maternal seed domain into the seed apoplasm surrounding the endosperm and embryo by poorly understood membrane transport processes(4-6). Plants are unique among eukaryotes in having specific P1B-ATPase pumps for the cellular export of zinc(7). In Arabidopsis, we show that two zinc transporting P1B-ATPases actively export zinc from the mother plant to the filial tissues. Mutant plants that lack both zinc pumps accumulate zinc in the seed coat and consequently have vastly reduced amounts of zinc inside the seed. Blockage of zinc transport was observed at both high and low external zinc supplies. The phenotype was determined by the mother plant and is thus due to a lack of zinc pump activity in the seed coat and not in the filial tissues. The finding that P1B-ATPases are one of the limiting factors controlling the amount of zinc inside a seed is an important step towards combating nutritional zinc deficiency worldwide.