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Cu+-chaperones are a diverse group of proteins that allocate Cu+ions to specific copper-proteins, creating different copper pools targeted to specific physiological processes. Symbiotic nitrogen fixation carried out in legume root nodules indirectly requires relatively large amounts of copper e.g. for energy delivery via respiration, for which targeted copper deliver systems would be required. MtNCC1 is a nodule-specific Cu+-chaperone encoded in theMedicago truncatulagenome, with a N-terminus Atx1-like domain that can bind Cu+with picomolar affinities. This gene is expressed primarily from the late infection zone to the early fixation zone, and is located in the cytosol, associated to plasma and symbiosome membranes, and within nuclei. Consistent with its key role in nitrogen fixation,ncc1mutants have a severe reduction of nitrogenase activity, and a 50% reduction in copper-dependent cytochromecoxidase activity. A subset of the copper-proteome is also affected in the mutant nodules. Many of these proteins can be pulled-down when using a Cu+-loaded N-terminal MtNCC1 moiety as a bait, indicating a role in nodule copper homeostasis and in copper-dependent physiological processes. Overall, these data suggest a pleiotropic role of MtNCC1 in copper delivery for symbiotic nitrogen fixation.

Journal of hazardous materials 442, 130062 (2023). doi:10.1016/j.jhazmat.2022.130062, Soybean (Glycine max (L.) Merr.) plants were exposed to various Cd concentrations from background and low non-toxic (0.5–50 nM) via sublethally toxic (< 550 nM) to highly, ultimately lethally toxic (3 µM) concentrations. Plants were cultivated hydroponically for 10 weeks until pod development stage of the control plants. The threshold and mechanism of sublethal Cd toxicity was investigated by metabolomics and metalloproteomics (HPLC-ICP-MS) measuring metal binding to proteins in the harvested roots. Spatial distribution of Cd was revealed by µXRF-CT. Specific binding of Cd to proteins already at 50 nM Cd revealed the likely high-affinity protein binding targets in roots, identified by protein purification from natural abundance. This revealed allantoinase, aquaporins, peroxidases and protein disulfide isomerase as the most likely high-affinity targets of Cd binding. Cd was deposited in cortex cell vacuoles at sublethal and bound to the cell walls of the outer cortex and the vascular bundle at lethal Cd. Cd binding to proteins likely inhibits them, and possibly induces detoxification mechanisms, as verified by metabolomics: allantoic acid and allantoate increased due to sublethal Cd toxicity. Changes of the Cd binding pattern indicated a detoxification strategy at lower Cd, but saturated binding sites at higher Cd concentrations., Published by Science Direct, New York, NY [u.a.]

SUMMARYBarium (Ba) and strontium (Sr) are often used as proxies for the reconstruction of past marine productivity and global climate. The ability to accumulate Ba2+ and Sr2+ in the form of crystals is rare among eukaryotes. Here we report that unicellular heterotrophs called diplonemids (Euglenozoa), one of the most abundant groups of marine planktonic protists, accumulate conspicuous amounts of these trace elements in the form of intracellular barite (BaSO4) and celestite (SrSO4) crystals, in concentrations greater than in other known Ba/Sr-accumulating organisms. Moreover, these flagellates can uptake Sr2+ exclusively or together with Ba2+ and form (Ba,Sr)SO4. One species, Namystinia karyoxenos, is naturally capable of intracellular accumulation of Ba2+ and Sr2+ 42,000 and 10,000 times relative to the surrounding medium. Altering the amounts of both elements in the medium resulted in corresponding changes in the quantity and composition of crystals. Planktonic copepods fed with diplonemids produce celestite-rich fecal pellets, which facilitate deposition of these minerals on the seafloor. We propose that diplonemids, which emerged during the Neoproterozoic era, qualify as impactful players of Ba2+/Sr2+ cycling in the ocean that possibly contributed to sedimentary rock formation over long geological periods.

The journal of experimental botany 71(4), 1628 - 1644 (2020). doi:10.1093/jxb/erz530, Solving the global environmental and agricultural problem of chronic low-level cadmium (Cd) exposure requires better mechanistic understanding. Here, soybean (Glycine max) plants were exposed to Cd concentrations ranging from 0.5 nM (background concentration, control) to 3 µM. Plants were cultivated hydroponically under non-nodulating conditions for 10 weeks. Toxicity symptoms, net photosynthetic oxygen production and photosynthesis biophysics (chlorophyll fluorescence: Kautsky and OJIP) were measured in young mature leaves. Cd binding to proteins [metalloproteomics by HPLC-inductively coupled plasma (ICP)-MS] and Cd ligands in light-harvesting complex II (LHCII) [X-ray absorption near edge structure (XANES)], and accumulation of elements, chloropyll, and metabolites were determined in leaves after harvest. A distinct threshold concentration of toxicity onset (140 nM) was apparent in strongly decreased growth, the switch-like pattern for nutrient uptake and metal accumulation, and photosynthetic fluorescence parameters such as Φ RE10 (OJIP) and saturation of the net photosynthetic oxygen release rate. XANES analyses of isolated LHCII revealed that Cd was bound to nitrogen or oxygen (and not sulfur) atoms. Nutrient deficiencies caused by inhibited uptake could be due to transporter blockage by Cd ions. The changes in specific fluorescence kinetic parameters indicate electrons not being transferred from PSII to PSI. Inhibition of photosynthesis combined with inhibition of root function could explain why amino acid and carbohydrate metabolism decreased in favour of molecules involved in Cd stress tolerance (e.g. antioxidative system and detoxifying ligands)., Published by Oxford Univ. Press, Oxford

Techniques for metal speciation analysis with subnanomolar (ppt) detection limits in complex matrices, with simultaneous quantification of matrix elements, have become a necessity for investigating targets of trace metal binding to macromolecules and pigments at environmentally relevant concentrations. In this work we optimized the analysis of such metal binding in a custom-built HPLC-ICP-MS system. Key elements of the optimization were the choice of components for the metal-free HPLC-DAD system and sector-field ICP-MS detection (ICF-sfMS) with desolvating injection and optimization of sample handling. Protein analysis was done using ammonium bicarbonate buffer and size exclusion chromatography (SEC-ICP-sfMS), with possible addition of anion exchange chromatography. Detection of metal exchange in pigments (chlorophylls and bacteriochlorophylls) was based on reversed-phase chromatography with a methanol-acetone gradient and coupling to the ICP-sfMS via a dedicated organic matrix interface (RPC-ICP-sfMS). The resulting HPLC-DAD-ICP-sfMS system has detection limits in the picomolar range in protein buffer, limited by the maximal achievable purity of buffers/solvents and not by system sensitivity. Tests for method optimization showed that sonication, meant to increase protein solubilization, leads to artifacts of metal loss from metalloproteins. Examples for Cd binding to soybean proteins and chlorophyll, Cr binding to Arabidopsis thaliana proteins, La binding to Desmodesmus quadricauda proteins, and Cu binding to Rhodospirillum rubrum proteins and pigments are shown. These application examples demonstrate that the system is sensitive enough to detect binding of metals to proteins and pigments at background concentration levels of typical nutrient solutions made from analytical grade chemicals, equivalent to ultratrace metal concentrations in nonpolluted environments.

Toxicity of lanthanides is generally regarded as low, and they even have been suggested to be beneficial at low concentrations. This research was conducted to investigate effects of Lanthanum (La) on Desmodesmus quadricauda, a freshwater green microalga. The algal cultures were treated with nanomolar La concentrations under controlled environmentally relevant conditions. Intracellular localization of La was analyzed with μXRF tomography in frozen-hydrated samples. At sublethal concentration (128 nM) La was in hotspots inside the cells, while at lethal 1387 nM that led to release of other ions (K, Zn) from the cells, La filled most of the cells. La had no clear positive effects on growth or photosynthetic parameters, but increasing concentrations led to a dramatic decrease in cell counts. Chlorophyll fluorescence kinetic measurements showed that La led to the inhibition of photosynthesis. Maximal photochemical quantum yield of the PSII reaction center in dark-adapted state (Fv/Fm) decreased at > 4.3 nM La during the 2nd week of treatment. Minimum dark-adapted fluorescence quantum yield (F0) increased at > 13.5 nM La during the 2nd week of treatment except for control (0.2 nM La, baseline from chemicals) and 0.3 nM La. NPQ at the beginning of the actinic light phase showed significant increase for all the treatments. Metalloproteomics by HPLC-ICPMS showed that La binds to a >500 kDa soluble protein complex already in the sub-nM range of La treatments, in the low nM range to a small-sized (3 kDa) soluble peptide, and at >100 nM La additionally binds to a 1.5 kDa ligand.

Background Many metals are essential for plants and humans. Knowledge of metal distribution in plant tissues in vivo contributes to the understanding of physiological mechanisms of metal uptake, accumulation and sequestration. For those studies, X-rays are a non-destructive tool, especially suited to study metals in plants. Results We present microfluorescence imaging of trace elements in living plants using a customized benchtop X-ray fluorescence machine. The system was optimized by additional detector shielding to minimize stray counts, and by a custom-made measuring chamber to ensure sample integrity. Protocols of data recording and analysis were optimised to minimise artefacts. We show that Zn distribution maps of whole leaves in high resolution are easily attainable in the hyperaccumulator Noccaea caerulescens. The sensitivity of the method was further shown by analysis of micro- (Cu, Ni, Fe, Zn) and macronutrients (Ca, K) in non-hyperaccumulating crop plants (soybean roots and pepper leaves), which could be obtained in high resolution for scan areas of several millimetres. This allows to study trace metal distribution in shoots and roots with a wide overview of the object, and thus avoids making conclusions based on singular features of tiny spots. The custom-made measuring chamber with continuous humidity and air supply coupled to devices for imaging chlorophyll fluorescence kinetic measurements enabled direct correlation of element distribution with photosynthesis. Leaf samples remained vital even after 20 h of X-ray measurements. Subtle changes in some of photosynthetic parameters in response to the X-ray radiation are discussed. Conclusions We show that using an optimized benchtop machine, with protocols for measurement and quantification tailored for plant analyses, trace metal distribution can be investigated in a reliable manner in intact, living plant leaves and roots. Zinc distribution maps showed higher accumulation in the tips and the veins of young leaves compared to the mesophyll tissue, while in the older leaves the distribution was more homogeneous.

In almost all photosynthetic organisms the photosynthetic pigments chlorophyll and bacteriochlorophyll (BChl) are Mg2+ containing complexes, but Mg2+ may be exchanged against other metal ions when these are present in toxic concentrations, leading to inactivation of photosynthesis. In this report we studied mechanisms of copper toxicity to the photosynthetic apparatus of Acidiphilium rubrum, an acidophilic purple bacterium that uses Zn2+ instead of Mg2+ as the central metal in the BChl molecules ([Zn]-BChl) of its reaction centres (RCs) and light harvesting proteins (LH1). We used a combination of in vivo measurements of photosynthetic activity (fast fluorescence and absorption kinetics) together with analysis of metal binding to pigments and pigment-protein complexes by HPLC-ICP-sfMS to monitor the effect of Cu2+ on photosynthesis of A. rubrum. Further, we found that its cytoplasmic pH is neutral. We compared these results with those obtained from Rhodospirillum rubrum, a purple bacterium for which we previously reported that the central Mg2+ of BChl can be replaced in vivo in the RCs by Cu2+ under environmentally realistic Cu2+ concentrations, leading to a strong inhibition of photosynthesis. Thus, we observed that A. rubrum is much more resistant to copper toxicity than R. rubrum. Only slight changes of photosynthetic parameters were observed in A. rubrum at copper concentrations that were severely inhibitory in R. rubrum and in A. rubrum no copper complexes of BChl were found. Altogether, the data suggest that [Zn]-BChl protects the photosynthetic apparatus of A. rubrum from detrimental insertion of Cu2+ (trans-metallation) into BChl molecules of its RCs.

Determination of trace element contents in crude oils constitutes a powerful geochemical tool, complementing organic geochemical analyses and allowing a deeper insight into the processes of oil generation, migration and maturation. Inductively coupled plasma mass spectrometry (ICP-MS) has been widely applied to trace element determination in petroleum samples due to its low detection limits and fast multi-element capability. However, quantification of several elements by quadrupole-based ICP-MS is hampered by low sensitivity due to high ionisation energy, severe polyatomic interferences and/or low abundance. In the present contribution an ICP tandem mass spectrometer (ICP-MS/MS), or triple quadrupole ICP-MS (ICP-QQQ-MS) has been used to overcome these limitations. Prior to analysis, samples were subjected to high pressure acid digestion, allowing external calibration by means of aqueous standards. 25 elements, including P, S, the first-row transition metals, As, Se, Re, Pb and U, could be quantified in one measurement. Polyatomic interferences on the lighter elements could be removed by reaction of the target ions with O 2 or NH 3 in the collision/reaction cell of the ICP-MS/MS. The accuracy of the analytical method was assessed by the analysis of the standard reference materials NIST SRM 1634c (trace elements in fuel oil) and 1084a (wear-metals in lubricating oil). The measurement procedure was applied to the analysis of crude oil samples from the Alpine Foreland Basin of Austria.

Magnesium (Mg2+) is the ubiquitous metal ion present in chlorophyll and bacteriochlorophyll (BChl), involved in photosystems in photosynthetic organisms. In the present study we investigated targets of toxic copper binding to the photosynthetic apparatus of the anoxygenic purple bacterium Rhodospirillum rubrum. This was done by a combination of in vivo measurements of flash photolysis and fast fluorescence kinetics combined with the analysis of metal binding to pigments and pigment-protein complexes isolated from Cu-stressed cells by HPLC-ICPMS (ICP-sfMS). This work concludes that R. rubrum is highly sensitive to Cu2+, with a strong inhibition of the photosynthetic reaction centres (RCs) already at 2 μM Cu2+. The inhibition of growth and of RC activity was related to the formation of Cu-containing BChl degradation products that occurred much more in the RC than in LH1. These results suggest that the shift of metal centres in BChl from Mg2+ to Cu2+ can occur in vivo in the RCs of R. rubrum under environmentally realistic Cu2+ concentrations, leading to a strong inhibition of the function of these RCs.

The use of transplanted moss (Pleurozium schreberi) in active biomonitoring of traffic-related emissions of Pd, Pt, and Rh was studied. Moss mats were transplanted to three locations along highway E75 (in Oulu, Finland) at three different distances from the highway. Five samples were collected from a background site after the same exposure period. Mass fractions of Pd, Pt, and Rh as well as mass fractions of 18 other elements were determined in these samples. The results indicated that P. schreberi is well suited for active biomonitoring of Pd, Pt, and Rh. Mass fractions above the background values were observed in the samples exposed to traffic-related emissions. When the results were compared with those of the other elements, high correlations of Pd, Pt, and Rh with commonly traffic-related elements (e.g., Cu, Ni, Sb, Zn, etc.) were found. It was also found that the amounts of Pd, Pt, and Rh in moss samples decreased when the distance to the highway increased. This trend gives evidence for the suitability of P. schreberi for active biomonitoring of Pd, Pt, and Rh. Furthermore, it can be concluded that the mass fractions determined in this study provide valuable evidence about the current state of Pd, Pt, and Rh emissions in Oulu, Finland.

Moss samples (Pleurozium schreberi) exposed to traffic-related emission of Pd, Pt and Rh were analysed in this study. Successful elimination of interferences was achieved in the determination of Pd, Pt and Rh mass fractions in these samples using inductively coupled plasma-tandem mass spectrometry (ICP-MS/MS). Based on the results, a reliable determination of Pd, Pt and Rh mass fractions in microwave-digested moss samples was obtained using ammonia (10% NH3 in He) in the collision/reaction cell when 103Rh was measured either on-mass (103→103) or with mass-shift (103→171) and mass-shifts for 108Pd (108→159) and 195Pt (195→229) were used. The ICP-MS/MS procedure was validated using BCR-723 (road dust) as a reference material. In addition, a good agreement between the ICP-MS/MS results and the results obtained with cloud point extraction and quadrupole ICP-MS was observed for the moss samples.

The non-marine Fushun Basin in NE China is a fault-controlled basin filled with Eocene sediments. It hosts the largest opencast coal and oil shale mine in Asia. A single thick oil shale layer overlying sub-bituminous coal occurs within the Middle Eocene Jijuntun Formation. Based on mineralogy, inorganic and organic geochemistry, organic petrography, stable isotope geochemistry, and vitrinite reflectance measurements, the depositional environment and the oil shale potential of the oil shale-bearing succession were investigated. The Jijuntun Formation is subdivided into a lower and an upper unit characterized by a low and high quality oil shale, respectively. The thick oil shale layer of the Jijuntun Formation developed under long-lasting stable conditions in a deep freshwater lake, after drowning of a swamp. The organic matter in the lower unit is characterized by landplant-derived macerals. The sediments containing a type II kerogen (HI: ∼400 mgHC/gTOC) were deposited during warm and humid conditions. Lacustrine organisms predominant in the upper unit are forming kerogen type I (HI: ∼700 mgHC/gTOC). High bioproductivity and excellent preservation conditions resulted in high TOC contents up to 23.6 wt.% in the upper unit. The organic matter preservation was controlled by photic zone anoxia originating in a temperature stratified water column in the deep lake, without significant changes in bottom water salinity. Mid-Eocene cooling during deposition of the upper unit of the Jijuntun Formation is reflected by clay mineral composition. A hot and arid climate favoring brackish conditions in a shallow lake prevailed during accumulation of the overlying carbonate-rich Xilutian Formation. Individual geochemical parameters in the Fushun Basin have to be used with caution, e.g. the maturity proxy Tmax is affected by kerogen type, the redox proxy Pr/Ph ratio is probably biased by different sources of isoprenoids. This demonstrates the importance of multi-proxy studies.

The incineration of municipal solid wastes is an important part of the waste management system along with recycling and waste disposal, and the solid residues produced after the thermal process have received attention for environmental concerns and the recovery of valuable metals. This study focuses on the Critical Raw Materials (CRM) content in solid residues from two Italian municipal waste incinerator (MSWI) plants. We sampled untreated bottom ash and fly ash residues, i.e. the two main outputs of common grate-furnace incinerators, and determined their total elemental composition with sensitive analytical techniques such as XRF and ICP-MS. After the removal of a few coarse metallic objects from bottom ashes, the corresponding ICP solutions were obtained using strong digestion methods, to ensure the dissolution of the most refractory components that could host significant amounts of precious metals and CRM. The integration of accurate chemical data with a substance flow analysis, which takes into account the mass balance and uncertainties assessment, indicates that bottom and fly ashes can be considered as a low concentration stream of precious and high-tech metals. The magnesium, copper, antimony and zinc contents are close to the corresponding values of a low-grade ore. The distribution of the elements flow between bottom and fly ash, and within different grain size fractions of bottom ash, is appraised. Most elements are enriched in the bottom ash flow, especially in the fine grained fractions. However, the calculated transfer coefficients indicate that Sb and Zn strongly partition into the fly ashes. The comparison with available studies indicates that the CRM concentrations in the untreated solid residues are comparable with those residues that undergo post-treatment beneficiations, e.g. separation between ferrous and non-ferrous fractions. The suggested separate collection of "fresh" bottom ash, which could be processed for further mineral upgrading, can constitute an attractive option of the waste management system, when physical-mechanical devices are not available or could not be implemented in old MSWI systems. The suggested procedure may lead to the improvement of recovery efficiency up to 83% for CRM and 94% for other valuable metals.

Bottom and fly ashes from Municipal Solid Waste Incinerators (MSWI) are hazardous products that present concern for their safe management. An attractive option to reduce their impact both on the environment and the financial commitment is turning MSWI ashes into secondary raw materials. In this study we present the REE content and distribution of bottom and fly ashes from MSWI after a highly effective digestion method and samples analysis by ICP-MS. The chondrite-normalised REE patterns of MSWI bottom and fly ash are comparable with that of crustal averages, suggesting a main geogenic source. Deviations from typical crustal pattern (e.g., Eu, Tb) disclose a contribution of likely anthropogenic provenance. The correlation with major elements indicates possible sources for REE and facilitates a preliminary resource assessment. Moreover, magnetic susceptibility measurements can be a useful prospecting method in urban ores made of MSWI ashes. The relationship between REE and some influencing parameters (e.g., Pricing Influence Factor) emphasises the importance of MSWI ash as alternative source of REE and the need of further efforts for REE recovery and purification from low concentrations but high flows waste.