Synchrotron X-rays reveal the modes of Fe binding and trace metal storage in the brown algae Laminaria digitata and Ectocarpus siliculosus

Funding Funding from the UK Natural Environment Research Council (NERC) through grants NE/D521522/1, NE/F012705/1, and Oceans 2025 (WP4.5) programs to FCK; the National Science Foundation (CHE-1664657) and the National Oceanic & Atmospheric Administration to CJC and FCK; and the MASTS pooling initiative (Marine Alliance for Science and Technology for Scotland, funded by the Scottish Funding Council and contributing institutions; grant reference HR09011) is gratefully acknowledged by FCK. PK would like to thank the European Commission for her postdoctoral fellowship (EC-Horizon 2020-MSCA-IF, grant no. 839151). AM and HK thank the Ministry of Education, Youth and Sports of the Czech Republic with co-financing from the European Union (grant "KOROLID", CZ.02.1.01/0.0/0.0/15_003/0000336) and the Czech Academy of Sciences (RVO: 60077344). AM, FK and HK are grateful for support from the European Community in the framework of the Access to Research Infrastructure Action of the Improving Human Potential Program to the ESRF (experiment LS-2772, beamline ID16AI). AM and HK thank Czech Government funding (Členství v European Synchrotron Radiation Facility, MŠMT – 33914/2017-1) supporting their work at the ESRF. GeoSoilEnviroCARS is supported by the National Science Foundation – Earth Sciences (EAR – 1634415) and Department of EnergyGeoSciences (DE-FG02-94ER14466). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Computational resources were supplied by the project "e-Infrastruktura CZ" (e-INFRA CZ LM2018140) supported by the Ministry of Education, Youth and Sports of the Czech Republic.Peer reviewedPublisher PD

Understanding the toxicity of arsenic, cadmium and copper in the model plant Ceratophyllum demersum using μXRF tomography and μ-XANES

Heavy metal uptake in plants is an important area not only for basic research, but also because of its impact on human nutrition and its application for the phytoremediation of contaminated soils. One of the most interesting topics in this area of research is the localisation and speciation of toxic metals&metalloids (e.g. As, Cd, Cu, Zn) inside the plant (e.g. [1], [2]). Here, we analyzed the distribution of non-hyperaccumulated As, Cd, Cu and Zn in the metal-sensitive shoot model plant Ceratophyllum demersum by µ XRF, and furthermore studied tissue-specific As speciation in the same sample using confocal µ XANES [3-5]. Samples of living leaves were prepared in capillaries, shock-frozen in supercooled isopentane, and maintained frozen-hydrated at about 100 K throughout the measurement. Elements in micro X-ray fluorescence (µ XRF) tomograms were quantified using standard-filled capillaries including a tomographic correction for X ray absorption in the sample. Measurements of As-stressed plants revealed that As was mainly sequestered in the epidermis. However, increasing As in the nutrient solution from 1 µM to 5 µM resulted in further increase of As in the vein and mesophyll but not in the epidermis of young leaves. Copper was mainly localized in the vein, and it did not change upon As exposure. In contrast, Zn was homogenously distributed over the whole leaf in control plants, but with increasing As, Zn was exported more towards the epidermis [3]. The in situ speciation of As in the same leaves was performed through µ XANES coupled to confocal detector optics. Thus we could distinguish As speciation at the tissue level i.e. epidermis, mesophyll and vein tissues (including xylem, phloem and separating tissue in between). The epidermis of a mature leaf contained the highest proportion of thiol-bound As (mostly with PCs), while in young leaves a lower proportion of As was thiol-bound. Further, at higher As concentrations the percentage of unbound AsIII increased in the vein and mesophyll of young leaves. Micro-XRF of Cd exposed plant leaves revealed changing distribution patterns of Cd and Zn at non-toxic (0.2 nM, 2 nM), moderately toxic (20 nM) and lethally toxic (200 nM) levels of Cd. Increasing Cd led to enhanced sequestration of Cd into non-photosynthetic tissues like epidermis and vein. At toxic Cd concentrations, Zn was redistributed and mainly found in the vein along with Cd, indicating an inhibition of their export from the vein [4]. At deficient and optimal copper supply, Cu was mainly localized in the vein. Copper deficiency did not alter the Cu distribution pattern, but lowered the tissue concentrations. In contrast, at toxic Cu after two weeks Cu was sequestered from veins towards the mesophyll and epidermis, with Cu concentrations in the epidermis reaching about half of the concentration in the vein. Longer treatment did not further increase the epidermal copper accumulation, but only Cu accumulation in the vein. Zn content of the leaves was reduced by increasing copper. Cu deficiency did not only increase Zn accumulation in the leaves, but also changed Zn distribution. While at optimal and toxic Cu, Zn was rather homogeneously distributed throughout the leaves, at deficient copper supply the additionally accumulated Zn was sequestered to the epidermis [5].References[1] H. Küpper, P.M.H. Kroneck, In: Metal Ions in Biological Systems, Volume 44, Chapter 5. (Eds: Sigel A, Sigel H, Sigel RKO). Marcel Dekker, Inc., New York; pp. 97-142 (2005)[2] E. Andresen, H. Küpper, Chapter 13, Volume 11 of series "Metal Ions in Life Sciences". (Eds: Sigel A, Sigel H, Sigel RKO). Springer Science + Business Media B.V., Dordrecht; pp. 395-414 (2013)[3] S. Mishra, G. Wellenreuther, J. Mattusch, H.-J. Stärk, H. Küpper, (in preparation for submission; 2013) [4] E. Andresen, J. Mattusch, G. Wellenreuther, G. Thomas, U.A. Abad, H. Küpper, (submitted to Metallomics, 2013)[5] G. Thomas, H.-J. Stärk,, G. Wellenreuther, Bryan C. Dickinson, H. Küpper, Aquatic toxicology, Early View: http://dx.doi.org/10.1016/j.aquatox.2013.05.008 (2013

Deficiency and toxicity of micronutrients and related elements in plants

Many heavy metals are essential trace elements, but elevated concentrations are toxic. One central focus of our research is on the response of higher plants and algae to trace metals in terms of uptake, transport, sequestration, complexation, deficiency, toxicity and detoxification. These processes are decisive factors in plant nutrition because of vastly different (due to natural and anthropogenic influence) trace metal concentrations in various habitats, ranging from deficient to toxic levels. In contrast to earlier studies in the field, we use conditions that allowed us to work also in the sub-nanomolar range and with a simulation of natural light- and temperature cycles. Thus, with the submerged water plant Ceratophyllum demersum as a model, we could show that heavy metal(loid) (As, Cd, Cr, Cu, Ni) concentrations that were previously considered as not having any effect actually have a strong impact on the plants, and with a different sequence of events than observed at very high concentrations. We used a combination of various biophysical and biochemical methods for measurements in vivo (e.g. photosynthesis biophysics, formation of reactive oxygen species, metal transport), in situ (e.g. quantitative (sub)cellular distribution and speciation of metals, mRNA levels) as well as on isolated proteins (for identification and characterisation of metalloproteins). For example, using metalloproteomics via native gels of protein extracts from plants that had been treated with heavy metals we are investigating the physiological and toxic binding of heavy metals to proteins. Analysis of pigments showed heavy metal-induced changes already at very low concentrations; this was reflected also by specific changes in biophysics of photosynthesis (e.g. spectral changes in non-photochemical quenching). As a result of the changes already mentioned, starch metabolism as well as production of reactive oxygen species were influenced by such sublethal concentrations of heavy metals and the metalloid arsenic in C. demersum. Our work in the sub-nanomolar range further showed that C. demersum stopped growth unless Cr(III) as Cr3+ or Cr(VI) as CrO42- became available, as extrapolated from the growth decrease towards the lowest achievable Cr (0.17 nM). This was a surprise because Cr is not regarded as an essential nutrient for plants. Chromium deficiency was furthermore found, although not at severe likely due to lower demands comparable to the lowest achievable Cr concentration, in the crop plants Glycine soja (soybean) and Triticum aestivum (wheat). We isolated several proteins that bind Cr with high affinity, and are currently characterizing them

Metal metabolism in plants - from the whole-plant to the molecular level with a focus on biochemistry and biophysics

Many heavy metals are essential micronutrients, and vastly different (with natural and anthropogenic causes) trace metal concentrations occur in various habitats, ranging from deficient to toxic levels. Therefore, one focus of plant research is on the response to trace metals in terms of uptake, transport, sequestration, speciation, deficiency, toxicity and detoxification. Early studies often used environmentally not relevant conditions, including too high metal concentrations and unrealistic light regimes. Further, individual processes often were not mechanistically interconnected, so that causes and consequences of metal effects remained unclear. In this talk, recent insights will be highlighted, mostly in the (sub-)nanomolar range of metal concentrations, with a simulation of natural light- and temperature cycles and trying to interconnect individual effects. The submerged water plant Ceratophyllum demersum turned out to be a useful shoot model, in which it could be shown that metal(loid) (As, Cd, Cr, Cu, Ni) concentrations that were previously considered as not having any effect actually have a strong impact on the plants, and with a different sequence of events than observed at very high concentrations. We used a combination of various biophysical and biochemical methods for measurements in vivo (e.g. photosynthesis biophysics, formation of reactive oxygen species, metal transport), in situ (e.g. quantitative (sub)cellular distribution and speciation of metals, mRNA levels) as well as on isolated proteins (for identification and characterization of metalloproteins). For example, using metalloproteomics via HPLC-ICPMS of protein extracts from plants that had been treated with heavy metals, changes in target sites of metal binding to proteins from deficient to toxic concentrations could be analyzed. Analysis of pigments, of metal(loid) sequestration and speciation showed clearly metal(loid)-induced changes already at very low concentrations. This was reflected also by specific alterations of photosynthesis biophysics. As a result, starch metabolism as well as production of reactive oxygen species were influenced

Transformation and Subcellular Distribution of Arsenic and Mechanism of its Sub-lethal Toxicity in Plants

Speciation of arsenic (As) in rice, the most As affected crop, and Ceratophyllum demersum, a good laboratorymodel for shoot, through chromatography and localization in the later through synchrotron based techniqueswas performed to explore the mechanism of As toxicity. In rice plant, exposed to inorganic arsenate (AsV) andmethylated As, most of the AsV and methylarsonate (MAV) were efficiently reduced to arsenite (AsIII) and MAIIIrespectively, but dimethylarsinate (DMAV) did not transformed. A large proportion of AsIII and MAIII were complexedwith thiols showing up to 20 and 16 As species respectively in the roots. Many of them were identifiedas new As-thiol species. Despite high complexation in root, more MA was translocated to shoot, with shoot/roottransfer factor being in order DMA>MA>inorganic As in rice.C. demersum, also displayed up to 60% accumulated As in the form of thiol complexed-AsIII. Most of which wasin epidermis of mature leaves as revealed by tissue resolved X-ray absorption near-edge spectroscopy (μ-XANES) of intact hydrated leaves. At sublethal concentration, As predominantly accumulated in the nucleus ofthe epidermal cells, as revealed by μ-X-ray fluorescence (μ-XRF), indicating replacement of P by As in DNA molecules,providing in vivo evidence for the proposed toxicity mechanism of AsV. While at lethal concentration,vacuole was the main storage site of As, yet a significant increase of unbound AsIII in mesophylls of young matureleaves occurred. This small amount of As reaching chloroplasts already caused a strong and specific inhibitionof tetrapyrrole biosynthesis and severe growth retardation.Taken together these results establish the mechanism of As toxicity and reported yield loss in paddy rice growingin As contaminated areas. Further, significant translocation of MAIII (more cytotoxic than AsIII) to rice shootscould also be an important factor inducing straighthead (spikelet sterility disorder) in rice

Speciation and Distribution of Arsenic in the Nonhyperaccumulator Macrophyte Ceratophyllum demersum

Although arsenic (As) is a common pollutant worldwide, many questions about As metabolism in nonhyperaccumulator plants remain. Concentration- and tissue-dependent speciation and distribution of As was analyzed in the aquatic plant Ceratophyllum demersum to understand As metabolism in nonhyperaccumulator plants. Speciation was analyzed chromatographically (high-performance liquid chromatography-[inductively coupled plasma-mass spectrometry]-[electrospray ionization-mass spectrometry]) in whole-plant extracts and by tissue-resolution confocal x-ray absorption near-edge spectroscopy in intact shock-frozen hydrated leaves, which were also used for analyzing cellular element distribution through x-ray fluorescence. Chromatography revealed up to 20 As-containing species binding more than 60% of accumulated As. Of these, eight were identified as thiol-bound (phytochelatins [PCs], glutathione, and cysteine) species, including three newly identified complexes: Cys-As(III)-PC2, Cys-As-(GS)2, and GS-As(III)-desgly-PC2. Confocal x-ray absorption near-edge spectroscopy showed arsenate, arsenite, As-(GS)3, and As-PCs with varying ratios in various tissues. The epidermis of mature leaves contained the highest proportion of thiol (mostly PC)-bound As, while in younger leaves, a lower proportion of As was thiol bound. At higher As concentrations, the percentage of unbound arsenite increased in the vein and mesophyll of young mature leaves. At the same time, x-ray fluorescence showed an increase of total As in the vein and mesophyll but not in the epidermis of young mature leaves, while this was reversed for zinc distribution. Thus, As toxicity was correlated with a change in As distribution pattern and As species rather than a general increase in many tissues

Different strategies of cadmium detoxification in the submerged macrophyte Ceratophyllum demersum L.

The heavy metal cadmium (Cd) is highly toxic to plants. To understand the mechanisms of tolerance and resistance to Cd, we treated the rootless, submerged macrophyte Ceratophyllum demersum L. with sub-micromolar concentrations of Cd under environmentally relevant conditions. X-ray fluorescence measurements revealed changing distribution patterns of Cd and Zn at non-toxic (0.2 nM, 2 nM), moderately toxic (20 nM) and highly toxic (200 nM) levels of Cd. Increasing Cd concentrations led to enhanced sequestration of Cd into non-photosynthetic tissues like epidermis and vein. At toxic Cd concentrations, Zn was redistributed and mainly found in the vein. Cd treatment induced the synthesis of phytochelatins (PCs) in the plants, with a threshold of induction already at 20 nM Cd for PC3. In comparison, in plants treated with Cu, elevated PC levels were detected only at the highest concentrations (100–200 nM Cu). Our results show that also non-accumulators like C. demersum store toxic metals in tissues where the heavy metal interferes least with metabolic pathways, but remaining toxicity interferes with micronutrient distribution. Furthermore, we found that the induction of phytochelatins is not proportional to metal concentration, but has a distinct threshold, specific for each PC species. Finally we could show that 20 nM Cd, which was previously regarded as non-toxic to most plants, already induces detoxifying mechanisms

MicroX-ray absorption near edge structure tomography reveals cell-specific changes of Zn ligands in leaves of turnip yellow mosaic virus infected plants

As many metals are essential for plants, excess or deficiency of them or alteration of their uptake, translocation, sequestration or physiological use can have severe consequences for plant growth and fitness. Therefore, investigating the distribution and speciation of metals in tissues is essential to understand plant physiology. We present a method based on non-destructive Synchrotron X-ray microtomography combined with microspectroscopy for studying metal distribution and speciation in plant tissues. By using the Maia detector system and the high flux of the undulator beam at the P06 beamline of the PETRA III synchrotron (at DESY), it was possible to record micro X-ray Absorption Near Edge Structure (μXANES) for every voxel of a tomogram. The metal coordination in regions of interest within the tissue samples could be determined by comparing the XANES with spectra of relevant reference compounds. Metal distribution and coordination were measured in shock frozen hydrated plant leaves in a cryostream, avoiding sample preparation artefacts like liquid cell content redistribution that occurs with other preparation methods, unequal distribution of stains in staining assays, sample degradation by beam damage and thawing, etc. A spatial resolution of 5 μm was selected, which is sufficient to resolve all leaf tissues (epidermis, palisade mesophyll, spongy mesophyll, veins), larger cells and biomineralization hotspots.As an application example, we studied the effect of infection with Turnip Yellow Mosaic Virus (TYMV) on the Zn distribution and the Zn speciation in duplicates of Noccaea ochroleucum. This non-accumulator plant grown with 100 μM Zn had enough metal to allow collecting significant spectroscopic data. We found that the TYMV infected samples formed biomineralization crystallites, showing strong spectroscopic similarity to Zn silicate

Poster P3: Toxicity and Detoxification of Cadmium in the Aquaticmacrophyte Ceratophyllum Demersum; session "Metal homeostasis and detoxification"

The heavy metal cadmium (Cd) is an important pollutant and poisonousto many organisms. We studied the effects of Cd on C.demersum under environmentally relevant conditions. High, moderateand low concentrations of Cd had different effects. Lethally toxicconcentrations (100–200 nM) led to growth stop and the plants’ability to perform photosynthesis (measured as Fv/Fm) decreased morethan twofold, consistent with decreased pigment content. Moderatelytoxic concentrations (10–50 nM) led to reduced growth, slightlyreduced pigment content, but hardly affected photosynthesis (measuredas O2 exchange and as Fv/Fm). Lower concentrations(0.2–5 nM) even had beneficial effects, like enhanced growth rate.When applied in low concentrations, Cd was homogeneously distributedin the whole cross-section of the leaves like a nutrient.Moderate and high Cd concentrations led to sequestration of Cd in thevascular bundle and the epidermis cells, where Cd does not affectphotosynthetic molecules. At toxic Cd concentrations, Zn was redistributedand mainly found in the vein along with Cd, indicatinginhibition of Zn transporters. Consistently, by metalloproteomics(HPLC-ICP-MS) we found that during Cd toxicity concentrations of Cdincreased in fractions of the major photosynthetic complexes while Mgdecreased, suggesting the replacement of Mg by Cd in chlorophylls.Furthermore, the induction of phytochelatins was not proportionalto metal concentration, but had distinct thresholds, specific for each PCspecies. PC3 especially was switch-like induced already at 20 nM Cd,which was previously regarded as non-toxic to most plants. Phytochelatinlevels at the lowest Cd concentrations were not detectable orbelow 0.1 % of the level at sublethally toxic concentrations, suggestingthat they do not have another function than metal detoxification