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Fungi are the leading cause of plant diseases worldwide and are responsible for enormous agricultural and industrial losses on a global scale. Cold plasma (CP) is a potential tool for eliminating or inactivating fungal contaminants from biological material such as seeds and grains. This study used a low-pressure radiofrequency CP system with oxygen as the feed gas to test the decontamination efficacy of different genera and species commonly colonising buckwheat grains. Two widely accepted methods for evaluating fungal decontamination after CP treatment of seeds were compared: direct cultivation technique or contamination rate method (%) and indirect cultivation or colony-forming units (CFU) method. For most of the tested fungal taxa, an efficient decrease in contamination levels with increasing CP treatment time was observed. Fusarium graminearum was the most susceptible to CP treatment, while Fusarium fujikuroi seems to be the most resistant. The observed doses of oxygen atoms needed for 1-log reduction range from 10 24 -10 25 m -2 . Although there was some minor discrepancy between the results obtained from both tested methods (especially in the case of Fusarium spp.), the trends were similar. The results indicate that the main factors affecting decontamination efficiency are spore shape, size, and colouration.

Increasing the concentration of an element in edible produce (i.e., biofortification) can mitigate the element deficiency in humans. Sprouts are small but popular part of healthy diets providing vitamins and essential elements throughout the year. Element composition of sprouts can easily be amended, e.g., by soaking the grains in element-rich solution before germination (grain-priming). In addition, pre-treatment of grains to improve element translocation from the solution into the grain may further enhance the element concentration in the sprout. Cold plasma technique could provide such solution, as it increases wettability and water uptake of grains. Grains of common buckwheat ( Fogopyrum esculentum Moench) were pre-treated/ untreated with cold plasma and soaked in ZnCl 2 solution/pure water. Germination tests, α-amylase activity, grain hydrophilic properties and water uptake were assessed. Element composition of grain tissues and of sprouts was assessed by micro-particle-induced-X-ray emission and X-ray fluorescence spectroscopy, respectively. Grain-priming increased Zn concentration in shoots of common buckwheat sprouts more than five-times, namely from 79 to 423 mg Zn kg -1 dry weight. Cold plasma treatment increased grain wettability and water uptake into the grain. However, cold plasma pre-treatment followed by grain-priming with ZnCl 2 did not increase Zn concentration in different grain tissues or in the sprouts more than the priming alone, but rather decreased the Zn concentration in sprout shoots (average ± standard error: 216 ± 6.13 and 174 ± 7.57 mg Zn kg -1 dry weight, respectively). When the fresh weight portion of whole sprouts (i.e., of roots and shoots) was considered, comparable average requirements of Zn, namely 24.5 % and 35 % for adult men and women would be satisfied by consuming cold plasma pre-treated and not pre-treated grains. Potential advantages of cold plasma pre-treatment need to be tested further, mainly to optimize the duration of soaking required to produce Zn-enriched sprouts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Starič, Remic, Vogel-Mikuš, Junkar, Vavpetič, Kelemen and Pongrac.)

Olive tree (Olea europaea L.) leaves have recently been recognised as a valuable source in cosmetic and pharmaceutical industry as well as in preparation of health-supporting beverages. Little is known about the element composition of olive leaves and almost nothing about tissue-specific allocation of elements. Element composition and tissue-specific distribution were determined in leaves of two olive cultivars, Leccino and Istarska bjelica using micro-particle induced X-ray emission (micro-PIXE). In leaves of the Istarska bjelica cultivar larger bulk concentrations of potassium, sodium, molybdenum and boron, but smaller concentrations of calcium and magnesium were found than in leaves of the Leccino cultivar. Tissue-specific investigation revealed that larger concentration of calcium in epidermis and in leaf blade tissues (secondary veins, palisade and spongy mesophyll) contributed to the larger leaf bulk calcium concentration in the Leccino cultivar. For magnesium, all leaf tissues, except the bundle sheath cells and consequently the main vascular bundle, contributed to the larger bulk concentration in the Leccino cultivar. Potassium was not predominant in any of the leaf tissues examined, while sodium and molybdenum were below the limit of detection, and boron not detectable by micro-PIXE. The results indicate that sinks for calcium and magnesium are stronger in specific leaf tissues of the Leccino than of the Istarska bjelica cultivar. The new understanding of tissue-specific allocation of elements in leaves of olive will serve as a basis for detailed studies into the effects of foliar and/or soil fertilisers in olive., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Arbuscular mycorrhizal fungi (AMF) exhibit great potential in heavy-metal immobilization in semi-aquatic habitats. Under high heavy-metal stress, however, the role of AMF is limited, and the detoxification mechanism of AMF in heavy metals' stabilization remains unclear. This study investigated the effects of AMF on a wetland plant (Iris pseudacorus) and chromium (Cr) immobilization at different water depths in semi-aquatic habitats with biochar addition. Results showed that AMF increased the physiological and photosynthetic functions in I. pseudacorus under Cr exposures. Besides, AMF alleviated the accumulation of reactive oxygen species and lipid peroxidation by enhancing the antioxidant enzyme activities. AMF and biochar significantly decreased Cr concentrations in outlet water and increased Cr accumulation in I. pseudacorus. Besides, biochar also vastly improved Cr accumulation in the substrate under the fluctuating water depth. AMF reduced Cr bioavailability in the substrate, with Cr (Ⅵ) concentrations and acid-soluble forms of Cr decreased by 0.3-64.5% and 19.0-40.8%, respectively. Micro-proton-induced X-ray emission was used to determine element localization and revealed that AMF improved the nutrients uptake by wetland plants and inhibited Cr translocation from roots to shoots. Overall, this study demonstrated that the interaction between AMF and biochar could significantly enhance the immobilization of high Cr concentrations in semi-aquatic habitats., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Cold plasma (CP) technology is a technique used to change chemical and morphological characteristics of the surface of various materials. It is a newly emerging technology in agriculture used for seed treatment with the potential of improving seed germination and yield of crops. Wheat seeds were treated with glow (direct) or afterglow (indirect) low-pressure radio-frequency oxygen plasma. Chemical characteristics of the seed surface were evaluated by XPS and FTIR analysis, changes in the morphology of the seed pericarp were analysed by SEM and AFM, and physiological characteristics of the seedlings were determined by germination tests, growth studies, and the evaluation of α-amylase activity. Changes in seed wettability were also studied, mainly in correlation with functionalization of the seed surface and oxidation of lipid molecules. Only prolonged direct CP treatment resulted in altered morphology of the seed pericarp and increased its roughness. The degree of functionalization is more evident in direct compared to indirect CP treatment. CP treatment slowed the germination of seedlings, decreased the activity of α-amylase in seeds after imbibition, and affected the root system of seedlings.

This study was undertaken to determine the effect of potassium silicate (K 2 SiO 3 ) on the physiological and growth characteristics and elemental composition of barley plants. Hydroponically grown barley ( Hordeum vulgare L.) var. Wilma was exposed to four different levels of Si in the form of K 2 SiO 3 at concentrations of 0 (Si 0 ), 0.5 (Si 0.5 ), 1 (Si 1 ) or 1.5 (Si 1.5 ) mM Si. Plants were analyzed for root length, number of dry leaves, number of trichomes, electron transport system activity in mitochondria (ETS), leaf pigment content and elemental composition of roots and leaves. Treatment with Si 0.5 significantly increased the concentration of total chlorophylls, root length and ETS activity in barley. Plants with no Si added to the nutrient solution had significantly more dry leaves than plants from all Si-treated groups. Necrosis was observed in Si 0 plants, while leaf damage was not visible in treated plants. According to the results of the study, we evidenced that plants were stressed due to Si deficiency. The addition of K 2 SiO 3 significantly affected the concentration of Si, K, Ca, Cl, S, Mn, Fe and Zn in roots and leaves of barley. In barley treated with Si 0.5 , plants showed the best performance in terms of their physiological characteristics and growth.

Buckwheat is an alternative crop known for its many beneficial effects on our health. Fungi are an important cause of plant diseases and food spoilage, often posing a threat to humans and animals. This study reports the effects of low-pressure cold plasma treatment on decontamination and germination of common (CB) and Tartary buckwheat (TB) grains. Both plasma glow and afterglow were applied. The glow treatment was more effective in decontamination: initial contamination was reduced to less than 30% in CB and 10% in TB. Fungal diversity was also affected as only a few genera persisted after the glow treatment; however, it also significantly reduced or even ceased the germination capacity of both buckwheat species. Detailed plasma characterisation by optical spectroscopy revealed extensive etching of outer layers as well as cotyledons. Afterglow treatment resulted in a lower reduction of initial fungal contamination (up to 30% in CB and up to 50% in TB) and had less impact on fungal diversity but did not drastically affect germination: 60-75% of grains still germinated even after few minutes of treatment. The vacuum conditions alone did not affect the fungal population or the germination despite an extensive release of water.

In plants, the response to stress, such as salinity, pathogen attack, drought, high concentration of metals, hyperthermia, and hypothermia, is usually accompanied by potassium ion (K + ) leakage from the cytosol to the cell wall, mediated by plasma membrane cation conductivity. Stress-induced electrolyte leakage co-occurs with accumulation of reactive oxygen species (ROS) and calcium ions (Ca 2+ ) and often results in programmed cell death (PCD). The development of X-ray and mass spectrometry (MS) based imaging techniques has enabled insight into the spatial tissue and cell-specific redistribution of major and trace elements during the stress response. In this chapter a workflow for sample preparation, imaging, and image analysis by X-ray and MS based techniques is presented., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The contribution of 1,8-dihydroxy naphthalene (DHN) melanin to cadmium (Cd) tolerance in two dark septate endophytes (DSE) of the genus Cadophora with different melanin content was investigated in vitro. The DSE isolate Cad#148 with higher melanin content showed higher tolerance to Cd than the less melanised Cad#149. Melanin synthesis was significantly reduced by Cd in both isolates with uninhibited melanin synthesis, in a dose-dependent manner. Inhibition of melanin synthesis by tricyclazole reduced the relative growth of Cad#148 exposed to Cd and did not affect Cad#149. Cd accumulation was not altered by tricyclazole in the two isolates, but it increased catalase and reduced glutathione reductase activity in more melanised Cad#148, indicating higher stress levels. In contrast, in Cad#149 the enzyme activity was less affected by tricyclazole, indicating a more pronounced role of melanin-independent Cd tolerance mechanisms. Cd ligand environment in fungal mycelia was analysed by extended EXAFS (X-ray absorption fine structure). It revealed that Cd was mainly bound to O- and S-ligands, including hydroxyl, carboxyl, phosphate and thiol groups. A similar proportion of S- and O- ligands (~35% and ~65%) were found in both isolates with uninhibited melanin synthesis. Among O-ligands two types with Cd-O-C- and Cd-O-P- coordination were identified. Tricyclazole altered Cd-O- ligand environment in both fungal isolates by reducing the proportion of Cd-O-C- and increasing the proportion of Cd-O-P coordination. DHN-melanin, among other tolerance mechanisms, significantly contributes to Cd tolerance in more melanised DSE fungi by immobilising Cd to hydroxyl groups and maintaining the integrity of the fungal cell wall., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Halophytes are able to tolerate relatively high concentrations of hazardous metals in a growing substrate, what makes them suitable candidates for phytoremediation of metal-contaminated soils. In this work, we aimed to study the physiological responses of the halophyte Sesuvium portulacastrum L. to Ni, with main focus on Ni localization, compartmentation and ligand environment, to decipher Ni tolerance and toxicity mechanisms. Seedlings were grown in hydroponic nutrient solution containing 0, 25, 50 and 100 μM Ni as NiCl 2 for 3 weeks. Ni localization in leaves was assessed by micro-proton-induced X-ray emission (micro-PIXE). Ni ligand environment was studied by Ni K-edge X-ray absorption near edge structure (XANES). In addition, Ni-soluble, weakly bound/exchangeable and insoluble leaf tissue fractions were determined by sequential extraction. Results show that S. portulacastrum is able to tolerate up to ~ 500 μg g -1 dry weight (DW) of Ni in the shoots without significant growth reduction. At higher Ni concentrations (> 50 μM Ni in nutrient solution), chloroses were observed due to the accumulation of Ni in photosynthetically active chlorenchyma as revealed by micro-PIXE. Water storage tissue represented the main pool for Ni storage. Incorporation of Ni into Ca-oxalate crystals was also observed in some specimens, conferring tolerance to high leaf Ni concentrations. The majority of Ni (> 70%) was found in soluble tissue fraction. Ni K XANES revealed Ni bound mainly to O- (55%) and N-ligands (45%). Ni toxicity at higher Ni levels was associated with Ni binding to amino groups of proteins in cytosol of chlorenchyma and increased level of lipid peroxidation. Proline levels also increased at high Ni exposures and were associated with Ni-induced oxidative stress and alteration of water regime.

This study used elemental and stable isotope composition to characterize Slovenian truffles and used multi-variate statistical analysis to classify truffles according to species and geographical origin. Despite the fact that the Slovenian truffles shared some similar characteristics with the samples originating from other countries, differences in the element concentrations suggest that respective truffle species may respond selectively to nutrients from a certain soil type under environmental and soil conditions. Cross-validation resulted in a 77% correct classification rate for determining the geographical origin and a 74% correct classification rate to discriminate between species. The critical parameters for geographical origin discriminations were Sr, Ba, V, Pb, Ni, Cr, Ba/Ca and Sr/Ca ratios, while from stable isotopes δ 18 O and δ 13 C values are the most important. The key variables that distinguish T.magnatum from other species are the levels of V and Zn and δ 15 N values. Tuber aestivum can be separated based on the levels of Ni, Cr, Mn, Mg, As, and Cu. This preliminary study indicates the possibility to differentiate truffles according to their variety and geographical origin and suggests widening the scope to include stable strontium isotopes., Competing Interests: The authors declare no conflict of interest.

The effects of foliar Se biofortification (Se+) of the lettuce on the transfer and toxicity of Hg from soil contaminated with HgCl 2 (H) and soil collected near the former Hg smelter in Idrija (I), to terrestrial food chain are explored, with Spanish slug as a primary consumer. Foliar application of Se significantly increased Se content in the lettuce, with no detected toxic effects. Mercury exerted toxic effects on plants, decreasing plant biomass, photochemical efficiency of the photosystem II (Fv/Fm) and the total chlorophyll content. Selenium biofortification (Se+ test group) had no effect on Hg bioaccumulation in plants. In slugs, different responses were observed in H and I groups; the I/Se+ subgroup was the most strongly affected by Hg toxicity, exhibiting lower biomass, feeding and growth rate and a higher hepatopancreas/ muscle Hg translocation, pointing to a higher Hg mobility in comparison to H group. Selenium increased Hg bioavailability for slugs, but with opposite physiological responses: alleviating stress in H/Se+ and inducing it in I/Se+ group, indicating different mechanisms of Hg-Se interactions in the food chain under HgCl 2 and Idrija soil exposures that can be mainly attributed to different Hg speciation and ligand environment in the soil., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This study provides information on mercury (Hg) localization, speciation and ligand environment in edible mushrooms: Boletus edulis, B. aereus and Scutiger pes-caprae collected at non-polluted and Hg polluted sites, by LA-ICP-MS, SR-μ-XRF and Hg L 3 -edge XANES and EXAFS. Mushrooms (especially young ones) collected at Hg polluted sites can contain more than 100 μg Hg g -1 of dry mass. Imaging of the element distribution shows that Hg accumulates mainly in the spore-forming part (hymenium) of the cap. Removal of hymenium before consumption can eliminate more than 50% of accumulated Hg. Mercury is mainly coordinated to di-thiols (43-82%), followed by di-selenols (13-35%) and tetra-thiols (12-20%). Mercury bioavailability, as determined by feeding the mushrooms to Spanish slugs (known metal bioindicators owing to accumulation of metals in their digestive gland), ranged from 4% (S. pes-caprae) to 30% (B. aereus), and decreased with increasing selenium (Se) levels in the mushrooms. Elevated Hg levels in mushrooms fed to the slugs induced toxic effects, but these effects were counteracted with increasing Se concentrations in the mushrooms, pointing to a protective role of Se against Hg toxicity through HgSe complexation. Nevertheless, consumption of the studied mushroom species from Hg polluted sites should be avoided., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

MeV-SIMS is an emerging mass spectrometry imaging method, which utilizes fast, heavy ions to desorb secondary molecules. High yields and low fragmentation rates of large molecules, associated with the electronic sputtering process, make it particularly useful in biomedical research, where insight into distribution of organic molecules is needed. Since the implementation of MeV-SIMS in to the micro-beam line at the tandem accelerator of Jožef Stefan Institute, MeV-SIMS provided some valuable observations on the distribution of biomolecules in plant tissue, as discussed by Jenčič et al. (Nucl. Inst. Methods Phys. Res. B. 371, 205-210, 2016; Nucl. Inst. Methods Phys. Res. B. 404, 140-145, 2017). However, limited focusing ability of the chlorine ion beam only allowed imaging at the tissue level. In order to surpass shortcomings of the existing method, we introduced a new approach, where we employ a continuous, low-current primary beam. In this mode, we bombard thin samples with a steady chlorine ion flux of approx. 5000 ions/s. After desorbing molecules, chlorine ions penetrate through the thinly cut sample and trigger the time-of-flight "start" signal on a continuous electron multiplier detector, positioned behind the sample. Such bombardment is more effective than previously used pulsing-beam mode, which demanded several orders of magnitude higher primary ion beam currents. Sub-micrometer focusing of low-current primary ion beam allows imaging of biological tissue on a subcellular scale. Simultaneously, new time-of-flight acquisition approach also improves mass resolution by a factor of 5. Within the article, we compare the performance of both methods and demonstrate the application of continuous mode on biological tissue. We also describe the thin sample preparation protocol, necessary for measurements with low primary ion currents.

In the past decade, the development of single particle-inductively coupled plasma mass spectrometry (SP-ICPMS) has revolutionized the field of nanometallomics. Besides differentiation between dissolved and particulate metal signals, SP-ICPMS can quantify the nanoparticle (NP) number concentration and size. Because SP-ICPMS is limited to characterization of NPs in solution, we show how solid sampling by laser ablation (LA) adds spatial-resolution characteristics for localized NP analysis in biomaterials. Using custom-made gelatin standards doped with dissolved gold and commercial or synthesized gold nanoparticles, LA-SP-ICPMS conditions such as laser fluence, beam size, and dwell time were optimized for NP analysis to minimize NP degradation, peak overlap, and interferences from dissolved gold. A data-processing algorithm to retrieve the NP number concentration and size was developed for this purpose. As a proof-of-concept, a sunflower-root-sample cross-section, originating from a sunflower plant exposed to gold NPs, was successfully imaged using the optimized LA-SP-ICPMS conditions for localized NP characterization.

Sesuvium portulacastrum, a halophyte with high tolerance to heavy metals like Cd, Pb and Ni is considered for phytoremediation of metal contaminated saline soils. The tolerance to a selected metal ion could, by hypothesis, be stimulated through in vitro adaptation and regeneration of the plant. Seedlings obtained by in vitro micro-propagation, were exposed to 0, 25 and 50 μM Ni, as NiCl 2 , in agar-based medium for 30 days. Growth parameters, plant water content, the concentration of photosynthetic pigments, proline and malondialdehyde (MDA) concentrations were determined. Nickel and nutrients distribution in leaves was studied by micro-Proton-Induced-X-ray-Emission (μ-PIXE). The results showed that Ni was mainly accumulated in vascular bundles, next in water storage tissues and chlorenchyma. Ni concentrations in chlorenchyma increased with increasing Ni in culturing medium, in direct relation to decrease of photosynthetic pigments and increase of oxidative stress. As compared to control plants, Ni induced substantial increase in MDA and proline accumulation. Plants exposed to 50 μM Ni accumulated up to 650 μg g -1 of Ni in the shoots, exhibiting chlorosis and necrosis and a drastically reduced plant growth. Perturbations in uptake and distribution of nutrients were observed, inducing mineral deficiency, probably through membrane leakage. The mineral nutrient disturbances induced by Ni could be highly implicated in the restriction of S. portulacastrum development under the acute 50 μM Ni level., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Proso millet (Panicum miliaceum L.) is an annual thermophilic, drought-resistant, short-season C4 grass that is suitable for growing under the present changing meteorological conditions. However, water shortage can reduce plant production and hinder mineral nutrition in plants, including silicon, which is of crucial importance for grasses. Ultraviolet radiation can mitigate the impacts of water shortage, and therefore we examined the effects of moderate water shortage and reduced ultraviolet radiation on different leaf traits, including leaf levels of silicon and other elements, and plant biomass production. Moderate water shortage and ambient ultraviolet radiation did not affect the contents of photosynthetic pigments, while they reduced stomata density. Water shortage significantly decreased leaf light reflectance in the ultraviolet and violet regions. Leaf silicon, calcium, phosphorus, and sulphur levels were significantly lower with reduced water availability and significantly higher with reduced ultraviolet radiation. Leaf silicon levels ranged from 1.5% to 2.5% of leaf dry mass, with leaf calcium levels from 0.3% to 0.6%. Except for chlorine and potassium, the levels of these elements in the leaves were significantly positively related (p ≤ 0.05) to soil moisture levels. Water availability, but not ultraviolet radiation, significantly reduced living leaf biomass.

This work aimed to study the role of arbuscular mycorrhizal fungi (AMF) in Hg and major mineral nutrient uptake and tissue localization of these elements in the roots of maize plants. Maize plants were grown in pots filled with non- and Hg-contaminated substrate (50 μg Hg g -1 as HgCl 2 ) and inoculated with two types of AMF inocula: a) Glomus sp. originating from Hg-polluted soil of a former Hg smelting site in Idrija, Slovenia, and b) commercial AM inoculum Symbivit. Controls were inoculated by corresponding bacterial extracts only. Tissue localization of Hg and major mineral nutrients was performed by laser ablation-inductively coupled plasma-mass spectroscopy (LA-ICP-MS) on cryofixed and freeze-dried root cross-sections. AMF colonization increased plant biomass in non-contaminated substrate, while this effect was not seen in Hg-contaminated substrate. Hg increased total plant biomass more than AMF inoculation, possibly through hormetic effects. AMF increased Hg uptake into the roots, as well as Hg transfer to the shoots. AMF affected plant mineral nutrient uptake, depending on the type of AMF inoculum and the presence of Hg. In the roots, Hg was mainly localized in rhizodermis and endodermis, followed by the cortex and the central cylinder. Higher Hg concentrations were detected in the central cylinder of AM plants than in that of the controls, pointing to a higher Hg mobility and potential bioavailability in AMF inoculated plants., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Two plant pathogenic fungi, Botrytis cinerea and Alternaria alternata, isolated from crop plants, were exposed to Cu in ionic (Cu 2+ ), microparticulate (MP, CuO) or nanoparticulate (NP, Cu or CuO) form, in solid and liquid culturing media in order to test fungal response and toxic effects of the mentioned compounds for the potential use as fungicides. B. cinerea has shown pronounced growth and lower levels of lipid peroxidation compared to A. alternata. Its higher resistance/tolerance is attributed mainly to biotransformation of CuO and Cu NPs and CuO MPs into a blue compound at the fungal/culturing media interface, recognized by Cu K-edge EXAFS analysis as Cu-oxalate complex. The pronounced activity of catechol-type siderophores and organic acid secretion in B. cinerea induce leaching and mobilization of Cu ions from the particles and their further complexation with extracellularly secreted oxalic acid. The ability of pathogenic fungus to biotransform CuO MPs and NPs hampers their use as fungicides. However the results show that B. cinerea has a potential to be used in degradation of Cu(O) nanoparticles in environment, copper extraction and purification techniques., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

UV radiation as an evolutionarily important environmental factor, significantly affects plants traits and alters the effects of other environmental factors. Single and combined effects of ambient UV radiation, its exclusion, and Se foliar treatments on Si concentrations and production of Si phytoliths in wheat (Triticum aestivum L.) cv. 'Reska' were studied. The effects of these treatments on growth parameters of the plants, structural and biochemical traits of the leaves, and interactions of the leaves with light, as Si incrustation is the first barrier to light at the leaf surface were also examined. Under ambient UV radiation and foliar treatment with 10mgL -1 sodium selenate solution, there was a trade-off between the plant investment in primary and secondary metabolism, as the production of UV-absorbing compounds was enhanced while photosynthetic pigment levels were reduced. Independent of Se treatment, ambient UV radiation lowered respiratory potential, Ca concentration, and leaf thickness, and increased Si concentration, Si phytoliths formation, and cuticle thickness. The Se treatment has little effect on plant traits and biomass production but it increased Se concentrations in the plants by >100-fold, independent of UV radiation. In combination with UV radiation Se strengthen the protection of plants against stress by increasing the amount of UV absorbing compounds, light reflectance and transmittance., (Copyright © 2016 Elsevier Inc. All rights reserved.)

It has been shown that halophytes are able to successfully cope with heavy metal toxicity, suggesting their possible use for remediation of metal contaminated soils. In this work, Ni tolerance and accumulation in two halophytes, Sesuvium portulacastrum (L.) L. and Cakile maritima Scop. was investigated. Seedlings of both species were subjected hydroponically during 21 days to 0, 25, 50, and 100 μM of NiCl 2 . The growth and photosynthesis parameters revealed that S. portulacastrum tolerates Ni better than C. maritima. The photosynthesis activity, chlorophyll content and photosystem II integrity were less impacted in Ni-treated S. portulacastrum as compared to C. maritima, although, Ni accumulated in higher concentrations in the shoots of S. portulacastrum (1050 μg g -1 DW) than in those of C. maritima (550 μg g -1 DW). The subcellular fractionation of Ni in the shoots of both species showed that C. maritima accumulated about 65% of Ni in the soluble fraction, while 28% was associated with the cell walls. In S. portulacastrum 44% of the total cellular Ni was seen in the soluble fraction and 43% was bound to the cell walls. It can be concluded that S. portulacastrum tolerates Ni better than C. maritima, most probably due to a better ability to sequester Ni in the cell walls, restricting its accumulation in the soluble fraction., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Exposure of beneficial soil organisms to chemical mixtures is of great concern and can result in unexpected deleterious consequences. We investigated the effects of concurrent soil contamination with monensin, a veterinary pharmaceutical and feed additive, and copper, on earthworm copper uptake and reproductive success. The animals were exposed for 14 or 28 days to both substances and the results showed that the Cu body burden of earthworms increases in the presence of monensin. The harmful effects of Cu on earthworm cocoon production were considerably higher when monensin was also present in the soil. To localise the copper in earthworm tissues, histological staining was performed using two different dyes (rubeanic acid and 5-4-(p-dimethylaminobenzylidene)-rhodanine). Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to quantify the Cu levels in the tissues. Cu was found predominantly in the gut wall. The Cu content in the body wall was at least ten times lower compared to the gut, but was proportional to the level of soil contamination. Concurrent soil contamination with monensin and copper resulted in higher earthworm Cu levels and in decreased reproductive success of these important soil decomposers., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This study aimed to relate the properties of incrusted plant tissues and structures as well as biomineral concentrations and localization with leaf reflectance and transmittance spectra from 280nm to 880nm in the grasses Phragmites australis, Phalaris arundinacea, Molinia caerulea and Deschampsia cespitosa, and the sedge Carex elata. Redundancy analysis revealed that prickle-hair length on adaxial surface and thickness of lower epidermis exerted significant effects in P. australis; prickle-hair density at abaxial leaf surface and thickness of epidermis on adaxial leaf surface in P. arundinacea; thickness of epidermis on adaxial leaf in D. cespitosa; prickle-hair density on adaxial leaf surface and thickness of cuticle in M. caerulea; and prickle-hair density on adaxial leaf surface and cuticle thickness of the lower side in C. elata. Micro-PIXE and LEXRF elemental localization analysis show that all of these structures and tissues are encrusted by Si and/or by Ca. Reflectance spectra were significantly affected by the Ca concentrations, while Si and Mg concentrations and the Ca concentrations significantly affected transmittance spectra. High concentrations of Mg were detected in epidermal vacuoles of P. arundinacea, M. caerulea and D. cespitosa. Al co-localises with Si in the cuticle, epidermis and/or prickle hairs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Main Conclusion: Wheat and its related genotypes show distinct distribution patterns for mineral nutrients in maternal and filial tissues in grains. X-ray-based imaging techniques are very informative to identify genotypes with contrasting tissue-specific localization of different elements. This can help in the selection of suitable genotypes for nutritional improvement of food grain crops. Understanding mineral localization in cereal grains is important for their nutritional improvement. Spatial distribution of mineral nutrients (Mg, P, S, K, Ca, Fe, Zn, Mn and Cu) was investigated between and within the maternal and filial tissues in grains of two wheat cultivars (Triticum aestivum Cv. WH291 and WL711), a landrace (T. aestivum L. IITR26) and a related wild species Aegilops kotschyi, using micro-proton-induced X-ray emission (µ-PIXE) and micro-X-ray fluorescence (µ-XRF). Aleurone and scutellum were major storage tissues for macro (P, K, Ca and Mg) as well as micro (Fe, Zn, Cu and Mn) nutrients. Distinct elemental distribution patterns were observed in each of the four genotypes. A. kotschyi, the wild relative of wheat and the landrace, T. aestivum L. IITR26, accumulated more Zn and Fe in scutellum and aleurone than the cultivated wheat varieties, WH291 and WL711. The landrace IITR26, accumulated far more S in grains, Mn in scutellum, aleurone and embryo region, Ca and Cu in aleurone and scutellum, and Mg, K and P in scutellum than the other genotypes. Unlike wheat, lower Mn and higher Fe, Cu and Zn concentrations were noticed in the pigment strand of A. kotschyi. Multivariate statistical analysis, performed on mineral distribution in major grain tissues (aleurone, scutellum, endosperm and embryo region) resolved the four genotypes into distinct clusters.

Silicon (Si) is an important structural element that can accumulate at high concentrations in grasses and sedges, and therefore Si structures might affect the optical properties of the leaves. To better understand the role of Si in light/leaf interactions in species rich in Si, we examined the total Si and silica phytoliths, the biochemical and morphological leaf properties, and the reflectance and transmittance spectra in grasses (Phragmites australis, Phalaris arundinacea, Molinia caerulea, Deschampsia cespitosa) and sedge (Carex elata). We show that these grasses contain >1% phytoliths per dry mass, while the sedge contains only 0.4%. The data reveal the variable leaf structures of these species and significant differences in the amount of Si and phytoliths between developing and mature leaves within each species and between grasses and sedge, with little difference seen among the grass species. Redundancy analysis shows the significant roles of the different near-surface silicified leaf structures (e.g., prickle hairs, cuticle, epidermis), phytoliths and Si contents, which explain the majority of the reflectance and transmittance spectra variability. The amount of explained variance differs between mature and developing leaves. The transmittance spectra are also significantly affected by chlorophyll a content and calcium levels in the leaf tissue., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Kosteletzkya virginica is a wetland halophyte that is a good candidate for rehabilitation of degraded salt marshes and production of oil as biodiesel. Salt marshes are frequently contaminated by heavy metals. The distribution of Zn in vegetative and reproductive organs of adult plants, and the NaCl influence on this distribution remain unknown and were thus explored in the present study. Plants were cultivated in a nutrient film technique system, from seedling stage until seed maturation in a control, Zn (100 μM), NaCl (50 mM) or Zn + NaCl medium. Photosynthesis, ion nutrition, malondialdehyde and non-protein thiol concentrations were quantified. Zinc distribution in reproductive organs was estimated by a laser ablation-inductively coupled plasma-mass spectrometry procedure (LA-ICP-MS). Adult plants accumulated up to 2 mg g(-1) DW Zn in the shoots. Zinc reduced plant growth, inhibited photosynthesis and reduced seed yield. Zinc accumulation in the seeds was only two times higher in Zn-treated plants than in controls. Exogenous NaCl neutralized the damaging action of Zn and modified the Zn distribution through a preferential accumulation of toxic ions in older leaves. Zinc was present in seed testa, endosperm and, to a lower extent, in embryo. Additional NaCl induced a chalazal retention of Zn during seed maturation and reduced final Zn seed content. It is concluded that NaCl 50 mM had a positive impact on the response of K. virginica to Zn toxicity and acts through a modification in Zn distribution rather than a decrease in Zn absorption.

Iron insufficiency is a worldwide problem in human diets. In cereals like wheat, the bran layer of the grains is an important source of iron. However, the dietary availability of iron in wheat flour is limited due to the loss of the iron-rich bran during milling and processing and the presence of anti-nutrients like phytic acid that keep iron strongly chelated in the grain. The present study investigated the localization of iron and phosphorus in grain tissues of wheat genotypes with contrasting grain iron content using synchrotron-based micro-X-ray fluorescence (micro-XRF) and micro-proton-induced X-ray emission (micro-PIXE). X-ray absorption near-edge spectroscopy (XANES) was employed to determine the proportion of divalent and trivalent forms of Fe in the grains. It revealed the abundance of oxygen, phosphorus, and sulphur in the local chemical environment of Fe in grains, as Fe-O-P-R and Fe-O-S-R coordination. Contrasting differences were noticed in tissue-specific relative localization of Fe, P, and S among the different genotypes, suggesting a possible effect of localization pattern on iron bioavailability. The current study reports the shift in iron distribution from maternal to filial tissues of grains during the evolution of wheat from its wild relatives to the present-day cultivated varieties, and thus suggests the value of detailed physical localization studies in varietal improvement programmes for food crops.

A LA-ICP-MS method based on a 213 nm Nd:YAG laser and a quadrupole ICP-MS has been developed for mapping of mercury in root cross-sections of maize (Zea mays L.) to investigate the mechanism of mercury uptake from soil and its potential translocation to the edible parts. Conventional rastering was found to be unusable due to sorption of mercury onto the internal parts of the LA device, giving rising to memory effects resulting in serious loss of resolution and inaccurate quantification. Spot analysis on a virtual grid on the surface of the root sections using washout times of 10s in between spots greatly alleviated problems related to these memory effects. By ablating straight through the root sections on a poly(methyl methacrylate) support the calibration process was simplified as internal standardization and matrix-matching could be circumvented. Mercury-spiked freeze-drying embedding medium, sectioned similarly to the root sections, was used for the preparation of the standards. Standards and root sections were subjected to spot analysis using the following operational parameters: beam diameter, 15 μm; laser fluence, 2.5 J cm(-2); repetition rate, 20 Hz; dwell time, 1s; acquisition time, 0.1s. The mercury peaks for standards and roots sections could be consistently integrated for quantification and construction of the 2D mercury maps for the root sections. This approach was successfully used to investigate the mercury distribution in root sections of maize grown in soil spiked to a level of 50 mg kg(-1) DW HgCl2. It was found that at given Hg concentrations in the substrate Hg ions practically do not cross root plasma membranes of the endodermal barrier, but are entirely retained in the root apoplastic space. This suggests that maize plants grown in Hg-contaminated areas translocate Hg to the upper edible parts of the plant only to a small extent., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Understanding the uptake, accumulation and distribution of toxic elements in plants is crucial to the design of effective phytoremediation strategies, especially in the case of complex multi-element pollution. Using micro-proton induced X-ray emission, the spatial distribution of Na, Mg, Al, Si, P, S, Cl, K, Ca, Mn, Fe, Zn, As, Br, Rb, Sr, Cd and Pb have been quantitatively resolved in roots and rhizomes of an obligate wetland plant species, Typha latifolia, treated with a mixture of 100 μM each of As, Cd and Pb, together. The highest concentrations of As, Cd and Pb were found in the roots of the T. latifolia, with tissue-specific distributions. The As was detected in the root rhizodermis, and in the rhizome the majority of the As was within the vascular tissues, which indicates the high mobility of As within T. latifolia. The Cd was detected in the root exodermis, and in the vascular bundle and epidermis of the rhizome. The highest Pb concentrations were detected in the root rhizodermis and exodermis, and in the epidermis of the rhizome. These data represent an essential step in the resolution of fundamental questions in plant ionomics., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A better understanding of the mechanisms that govern copper (Cu) uptake, distribution and tolerance in Brassica carinata plants in the presence of chelators is needed before significant progress in chelate-assisted Cu phytoextraction can be made. The aims of this study were therefore to characterise (S,S)-N,N'-ethylenediamine disuccinic acid (EDDS)-assisted Cu uptake, and to compare the spatial distribution patterns of Cu in the roots and leaves of B. carinata plants. The plants were treated with 30 μM or 150 μM CuSO(4) or CuEDDS in hydroponic solution. Quantitative Cu distribution maps and concentration profiles across root and leaf cross-sections of the desorbed plants were obtained by micro-proton induced X-ray emission. In roots, the 30 μM treatments with both CuSO(4) and CuEDDS resulted in higher Cu concentrations in epidermal/cortical regions. At 150 μM CuSO(4), Cu was mainly accumulated in root vascular bundles, whereas with 150 μM CuEDDS, Cu was detected in endodermis and the adjacent inner cortical cell layer. Under all treatments, except with a H(+)-ATP-ase inhibitor, the Cu in leaves was localised mainly in vascular tissues. The incubation of plants with 150 μM CuEDDS enhanced metal translocation to shoots, in comparison to the corresponding CuSO(4) treatment. Inhibition of H(+)-ATPase activity resulted in reduced Cu accumulation in 30 μM CuEDDS-treated roots and 150 μM CuEDDS-treated leaves, and induced changes in Cu distribution in the leaves. This indicates that active mechanisms are involved in retaining Cu in the leaf vascular tissues, which prevent its transport to photosynthetically active tissues. The physiological significance of EDDS-assisted Cu uptake is discussed., (Copyright © 2012 Elsevier B.V. All rights reserved.)