Symplasmic and transmembrane zinc transport is modulated by cadmium in the Cd/Zn hyperaccumulator Sedum alfredii.

This study investigated the influence of Cd (25 µM) on Zn accumulation in a hyperaccumulating (HE) and a non-hyperaccumulating (NHE) ecotype of Sedum alfredii Hance at short-term supply of replete (Zn5, 5 µM) and excess (Zn400, 400 µM) Zn. Cd inhibited Zn accumulation in both ecotypes, especially under Zn400, in organs with active metal sequestration, i.e. roots of NHE and shoots of HE. Direct biochemical Cd/Zn competition at the metal-protein interaction and changes in transporter gene expression contributed to the observed accumulation patterns in the roots. Specifically, in HE, Cd stimulated SaZIP4 and SaPCR2 under Zn5, but downregulated SaIRT1 and SaZIP4 under Zn400. However, Cd downregulated related transporter genes, except for SaNRAMP1, in NHE, irrespective of Zn. Cadmium stimulated casparian strip (CSs) development in NHE, as part of the defense response, while it had a subtle effect on the (CS) in HE. Moreover, Cd delayed the initiation of the suberin lamellae (SL) in HE, but stimulated SL deposition in NHE under both Zn5 or Zn400. Changes in suberization were mainly ascribed to suberin-biosynthesis-related genes and hormonal signaling. Altogether, Cd regulated Zn accumulation mainly via symplasmic and transmembrane transport in HE, while Cd inhibited both symplasmic and apoplasmic Zn transport in NHE.

Zinc priming enhances Capsicum annuum immunity against infection by Botrytis cinerea- From the whole plant to the molecular level.

Micronutrient manipulation can enhance crop resilience against pathogens, but the mechanisms are mostly unknown. We tested whether priming Capsicum annuum plants with zinc (5 µM Zn) or manganese (3 µM Mn) for six weeks increases their immunity against the generalist necrotroph Botrytis cinerea compared to deficient (0.1 µM Zn, 0.02 µM Mn) and control conditions (1 µM Zn, 0.6 µM Mn). Zinc priming reduced the pathogen biomass and lesion area and preserved CO2 assimilation and stomatal conductance. Zinc mobilization at the infection site, visualized by micro-X-ray fluorescence, was accompanied by increased Zn protein binding obtained by size exclusion HPLC-ICP/MS. A common metabolic response to fungal infection in Zn- and Mn-primed plants was an accumulation of corchorifatty acid F, a signaling compound, and the antifungal compound acetophenone. In vitro tests showed that the binding of Zn2+ increased, while Mn2+ binding decreased acetophenone toxicity against B. cinerea at concentrations far below the toxicity thresholds of both metals in unbound (aquo complex) form. The metal-specific response to fungal infection included the accumulation of phenolics and amino acids (Mn), and the ligand isocitrate (Zn). The results highlight the importance of Zn for pepper immunity through direct involvement in immunity-related proteins and low molecular weight Zn-complexes, while Mn priming was inefficient.

Acclimation to medium-level non-lethal iron limitation: Adjustment of electron flow around the PSII and metalloprotein expression in Trichodesmium erythraeum IMS101.

The aim of this study was to investigate how acclimation to medium-level, long-term, non-lethal iron limitation changes the electron flux around the Photosystem II of the oceanic diazotroph Trichodesmium erythraeum IMS101. Fe availability of about 5× and 100× lower than a replete level, i.e. conditions common in the natural environment of this cyanobacterium, were applied in chemostats. The response of the cells was studied not only in terms of growth, but also mechanistically, measuring the chlorophyll fluorescence of dark-adapted filaments via imaging fluorescence kinetic microscopy (FKM) with 0.3 ms time resolution. Combining these measurements with those of metal binding to proteins via online coupling of metal-free HPLC (size exclusion chromatography SEC) to sector-field ICP-MS allowed to track the fate of the photosystems, together with other metalloproteins. General increase of fluorescence has been observed, with the consequent decrease in the quantum yields φ of the PSII, while the efficiency ψ of the electron flux between PSII and the PSI remained surprisingly unchanged. This indicates the ability of Trichodesmium to cope with a situation that makes assembling the many iron clusters in Photosystem I a particular challenge, as shown by decreasing ratios of Fe to Mg in these proteins. The negative effect of Fe limitation on PSII may also be due to its fast turnover. A broader view was obtained from metalloproteomics via HPLC-ICP-MS, revealing a differential protein expression pattern under iron limitation with a drastic down-regulation especially of iron-containing proteins and some increase in low MW metal-binding complexes.

Copper and zinc accumulation, distribution, and tolerance in Pistia stratiotes L.; revealing the role of root caps.

Pollution by potentially toxic trace metals, such as copper or zinc, is global. Both Cu and Zn are essential microelements, which in higher concentrations become toxic. The aquatic plant Pistia stratiotes(L. has great potential for phytoremediation. Also, it has an unusually large and easily detachable root cap, which makes it a suitable model for studying the potential role of the root cap in metal uptake. Plant response to environmentally relevant concentrations of Cu (0.1, 0.3, and 1 µM) and Zn (0.3, 1, and 3 µM) was investigated with the aim of studying their interaction and distribution at the root tissue level as well as revealing their tolerance mechanisms. Changes in the root anatomy and plant ionome were determined using light and fluorescence microscopy, ICP-MS, and µXRF imaging. Alterations in photosynthetic activity caused by Cu or Zn excesses were monitored by direct imaging of fast chlorophyll fluorescence kinetics (OJIP). Fe and Mn were preferentially localized in the root cap, while Ca, Cu, Ni, and Zn were mainly in the root tip regardless of the Cu/Zn treatment. Translocation of Cu and Zn to the leaves increased with higher doses, however the translocation factor was the lowest in the highest treatments. Measurements of photosynthetic parameters showed a higher susceptibility of electron transport flux from QA to QB under increasing Cu than Zn supply. This, along with our findings regarding the root anatomy and the differences in Ca accumulation and distribution, led to the conclusion that P. stratiotes is more effective for Zn remediation than Cu.

Cadmium and Zn hyperaccumulation provide efficient constitutive defense against Turnip yellow mosaic virus infection in Noccaea caerulescens.

To understand the role of Zn and Cd in anti-viral defence, Zn/Cd hyperaccumulator Noccaea caerulescens plants grown with deficient (0.3 µM), replete (10 µM) and excess (100 µM) Zn2+ and Cd (10 µM Zn2+ + 1 µM Cd2+) were infected with Turnip yellow mosaic virus (TYMV). Gas exchange and chlorophyll fluorescence kinetics analyses demonstrated direct TYMV effects on photosynthetic light reactions but N. caerulescens was more resistant against TYMV than the previously studied non-hyperaccumulator N. ochroleucum. Virus abundance and photosynthesis inhibition were the lowest in the high Zn and Cd treatments. RNAseq analysis of 10 µM Zn2+ plants revealed TYMV-induced upregulation of Ca transporters, chloroplastic ZTP29 and defence genes, but none of those that are known to be strongly involved in hyperaccumulation. Synchrotron µ-XRF tomography, however, showed that Zn hyperaccumulation remained strongest in vacuoles of epidermal storage cells regardless of infection. This was in contrast to N. ochroleucum, where apoplastic Zn drastically increased in response to TYMV. These results suggest that the antiviral response of N. caerulescens is less induced by the onset of this biotic stress, but it is rather a permanent resistant state of the plant. Real-time qPCR revealed upregulation of ferritin in Zn10 infected plants, suggesting Fe deprivation as a virus defence strategy under suboptimal Zn supply.

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

Iron is accumulated symplastically in kelp in a non-ferritin core that seems to be a general feature of brown algae. Microprobe studies show that Fe binding depends on tissue type. The sea is generally an iron-poor environment and brown algae were recognized in recent years for having a unique, ferritin-free iron storage system. Kelp (Laminaria digitata) and the filamentous brown alga Ectocarpus siliculosus were investigated using X-ray microprobe imaging and nanoprobe X-ray fluorescence tomography to explore the localization of iron, arsenic, strontium, and zinc, and micro-X-ray absorption near-edge structure (µXANES) to study Fe binding. Fe distribution in frozen hydrated environmental samples of both algae shows higher accumulation in the cortex with symplastic subcellular localization. This should be seen in the context of recent ultrastructural insight by cryofixation-freeze substitution that found a new type of cisternae that may have a storage function but differs from the apoplastic Fe accumulation found by conventional chemical fixation. Zn distribution co-localizes with Fe in E. siliculosus, whereas it is chiefly located in the L. digitata medulla, which is similar to As and Sr. Both As and Sr are mostly found at the cell wall of both algae. XANES spectra indicate that Fe in L. digitata is stored in a mineral non-ferritin core, due to the lack of ferritin-encoding genes. We show that the L. digitata cortex contains mostly a ferritin-like mineral, while the meristoderm may include an additional component.

Effect of nanomolar concentrations of lanthanum on Desmodesmus quadricauda cultivated under environmentally relevant conditions.

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.

Interactions between zinc and Phomopsis longicolla infection in roots of Glycine max.

Phomopsis. longicolla is a hemibiotrophic fungus causing significant soybean yield loss worldwide. To reveal the role of zinc in plant-pathogen interactions, soybean seedlings were grown hydroponically with a range of Zn concentrations, 0.06 µM (deficient, Zn0), 0.4 µM (optimal growth), 1.5 µM, 4 µM, 12 µM, and toxic 38 µM, and were subsequently inoculated with P. longicolla via the roots. In vivo analysis of metal distribution in tissues by micro-X-ray fluorescence showed local Zn mobilization in the root maturation zone in all treatments. Decreased root and pod biomass, and photosynthetic performance in infected plants treated with 0.4 µM Zn were accompanied with accumulation of Zn, jasmonoyl-L-isoleucine (JA-Ile), jasmonic acid, and cell wall-bound syringic acid (cwSyA) in roots. Zn concentration in roots of infected plants treated with 1.5 µM Zn was seven-fold higher than in the 0.4 µM Zn treatment, which together with accumulation of JA-Ile, cwSyA, cell wall-bound vanilic acid and leaf jasmonates contributed to maintaining photosynthesis and pod biomass. Host-pathogen nutrient competition and phenolics accumulation limited the infection in Zn-deficient plants. The low infection rate in Zn 4 µM-treated roots correlated with salicylic and 4-hydroxybenzoic acid, and cell wall-bound p-coumaric acid accumulation. Zn toxicity promoted pathogen invasion and depleted cell wall-bound phenolics. The results show that manipulation of Zn availability improves soybean resistance to P. longicolla by stimulating phenolics biosynthesis and stress-inducible phytohormones.

The Medicago truncatula Yellow Stripe1-Like3 gene is involved in vascular delivery of transition metals to root nodules.

Symbiotic nitrogen fixation carried out in legume root nodules requires transition metals. These nutrients are delivered by the host plant to the endosymbiotic nitrogen-fixing bacteria living within the nodule cells, a process in which vascular transport is essential. As members of the Yellow Stripe-Like (YSL) family of metal transporters are involved in root to shoot transport, they should also be required for root to nodule metal delivery. The genome of the model legume Medicago truncatula encodes eight YSL proteins, four of them with a high degree of similarity to Arabidopsis thaliana YSLs involved in long-distance metal trafficking. Among them, MtYSL3 is a plasma membrane protein expressed by vascular cells in roots and nodules and by cortical nodule cells. Reducing the expression level of this gene had no major effect on plant physiology when assimilable nitrogen was provided in the nutrient solution. However, nodule functioning was severely impaired, with a significant reduction of nitrogen fixation capabilities. Further, iron and zinc accumulation and distribution changed. Iron was retained in the apical region of the nodule, while zinc became strongly accumulated in the nodule veins in the ysl3 mutant. These data suggest a role for MtYSL3 in vascular delivery of iron and zinc to symbiotic nitrogen fixation.

Interaction Between Zn Deficiency, Toxicity and Turnip Yellow Mosaic Virus Infection in Noccaea ochroleucum.

Zinc is essential for the functioning of numerous proteins in plants. To investigate how Zn homeostasis interacts with virus infection, Zn-tolerant Noccaea ochroleucum plants exposed to deficient (Zn'0'), optimal (Zn10), and excess Zn (Zn100) concentrations, as well as Cd amendment, were infected with Turnip yellow mosaic virus (TYMV). Imaging analysis of fluorescence kinetics from the µs (OJIP) to the minutes (Kautsky effect, quenching analysis) time domain revealed strong patchiness of systemic virus-induced photosystem II (PSII) inhibition. That was more pronounced in Zn-deficient plants, while Zn excess acted synergistically with TYMV, in both cases resulting in reduced PSII reaction centers. Infected Cd-treated plants, already severely stressed, showed inhibited non-photochemical quenching and PSII activity. Quantitative in situ hybridization at the cellular level showed increased gene expression of ZNT5 and downregulation of HMA4 in infected Zn-deficient leaves. In Zn10 and Zn100 infected leaves, vacuolar sequestration of Zn increased by activation of HMA3 (mesophyll) and MTP1 (epidermis). This correlated with Zn accumulation in the mesophyll and formation of biomineralization dots in the cell wall (Zn100) visible by micro X-ray fluorescence tomography. The study reveals the importance of adequate Zn supply and distribution in the maintenance of photosynthesis under TYMV infection, achieved by tissue-targeted activation of metal transporter gene expression.

Analysis of trace metal distribution in plants with lab-based microscopic X-ray fluorescence imaging.

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.

A secreted fungal histidine- and alanine-rich protein regulates metal ion homeostasis and oxidative stress.

Like pathogens, beneficial endophytic fungi secrete effector proteins to promote plant colonization, for example, through perturbation of host immunity. The genome of the root endophyte Serendipita indica encodes a novel family of highly similar, small alanine- and histidine-rich proteins, whose functions remain unknown. Members of this protein family carry an N-terminal signal peptide and a conserved C-terminal DELD motif. Here we report on the functional characterization of the plant-responsive DELD family protein Dld1 using a combination of structural, biochemical, biophysical and cytological analyses. The crystal structure of Dld1 shows an unusual, monomeric histidine zipper consisting of two antiparallel coiled-coil helices. Similar to other histidine-rich proteins, Dld1 displays varying affinity to different transition metal ions and undergoes metal ion- and pH-dependent unfolding. Transient expression of mCherry-tagged Dld1 in barley leaf and root tissue suggests that Dld1 localizes to the plant cell wall and accumulates at cell wall appositions during fungal penetration. Moreover, recombinant Dld1 enhances barley root colonization by S. indica, and inhibits H2 O2 -mediated radical polymerization of 3,3'-diaminobenzidine. Our data suggest that Dld1 has the potential to enhance micronutrient accessibility for the fungus and to interfere with oxidative stress and reactive oxygen species homeostasis to facilitate host colonization.

Chronic exposure of soybean plants to nanomolar cadmium reveals specific additional high-affinity targets of cadmium toxicity.

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).

X-ray absorption spectroscopy of an investigational anticancer gallium(III) drug: interaction with serum proteins, elemental distribution pattern, and coordination of the compound in tissue.

Tris(8-quinolinolato)gallium(III) (1, KP46) is a very promising investigational anticancer drug. Its interaction with serum proteins, elemental distribution, and coordination in tissue were investigated with X-ray absorption (XAS) methods. Model compounds with mixed O, N, and/or S donor atoms are reported. The coordination and structure of 1 in cell culture medium (minimum essential medium, MEM) and fetal calf serum (FCS) were probed by XANES and EXAFS. The interaction of 1 with the serum proteins apotransferrin (apoTf) and human serum albumin (HSA) was addressed as well. By application of micro-XAS to tissue samples from mice treated with 1, the gallium distribution pattern was analyzed and compared to those of physiological trace elements. The complex 1 turned out to be very stable under physiological conditions, in cell culture media and in tissue samples. The coordination environment of the metal center remains intact in the presence of apoTf and HSA. The gallium distribution pattern in tumor and liver tissue revealed high similarities to the distribution patterns of Zn and Fe, minor similarities to Cu and Ni, and no similarity to Ca.

Protonation of the oxygen axial ligand in galactose oxidase model compounds as seen with high resolution X-ray emission experiments and FEFF simulations.

X-ray Emission Spectroscopy (XES) crossover peaks were shown to be sensitive to the protonation state of solvent molecules in the Zn protein carbonic anhydrase and its model compounds. Here we extend such studies to galactose oxidase models i.e. Cu(ii) open d-shell systems, illustrating that XES combined with FEFF8 simulations reflect changes in the protonation state of the phenolate ligand for the copper center.

Functional groups and sulfur K-edge XANES spectra: divalent sulfur and disulfides.

Sulfur K-edge XANES was measured for two divalent sulfurs (dibenzyl and benzyl phenyl) and two disulfides (dibenzyl and diphenyl). The absorption spectra could be assigned using density functional theory with the "half core hole" approximation for the core hole including relaxation of selected excited states at the absorption edge. Analysis of the molecular orbitals shows that the characteristic double peak of the dibenzyl disulfide arises as a consequence of the enhanced splitting of the LUMO (lowest unoccupied molecular orbital) and the LUMO + 1. Exchange of the ligand benzyl by phenyl introduces more transitions at the absorption edge, which enhance the broadening in the divalent sulfur and splitting of the peaks of the disulfide. It is shown that different ligand groups introduce significant differences in the absorption edge, which poses a problem for the speciation analysis when the ligand groups are not clearly defined.

New reduction pathways for ctc-[PtCl2(CH3CO2)2(NH3)(Am)] anticancer prodrugs.

Reduction of anticancer prodrugs such as ctc-[PtCl(2)(CH(3)CO(2))(2)(NH(3))(Am)] can yield three products in addition to the expected cis-[PtCl(2)(NH(3))(Am)]. A possible explanation is that reduction proceeds by several pathways where in addition to the loss of two axial ligands, one axial (acetato) and one equatorial (chlorido) ligand, or two equatorial ligands are eliminated.

Complexation and toxicity of copper in higher plants. II. Different mechanisms for copper versus cadmium detoxification in the copper-sensitive cadmium/zinc hyperaccumulator Thlaspi caerulescens (Ganges Ecotype).

The cadmium/zinc hyperaccumulator Thlaspi caerulescens is sensitive toward copper (Cu) toxicity, which is a problem for phytoremediation of soils with mixed contamination. Cu levels in T. caerulescens grown with 10 microm Cu(2+) remained in the nonaccumulator range (<50 ppm), and most individuals were as sensitive toward Cu as the related nonaccumulator Thlaspi fendleri. Obviously, hyperaccumulation and metal resistance are highly metal specific. Cu-induced inhibition of photosynthesis followed the "sun reaction" type of damage, with inhibition of the photosystem II reaction center charge separation and the water-splitting complex. A few individuals of T. caerulescens were more Cu resistant. Compared with Cu-sensitive individuals, they recovered faster from inhibition, at least partially by enhanced repair of chlorophyll-protein complexes but not by exclusion, since the content of Cu in their shoots was increased by about 25%. Extended x-ray absorption fine structure (EXAFS) measurements on frozen-hydrated leaf samples revealed that a large proportion of Cu in T. caerulescens is bound by sulfur ligands. This is in contrast to the known binding environment of cadmium and zinc in the same species, which is dominated by oxygen ligands. Clearly, hyperaccumulators detoxify hyperaccumulated metals differently compared with nonaccumulated metals. Furthermore, strong features in the Cu-EXAFS spectra ascribed to metal-metal contributions were found, in particular in the Cu-resistant specimens. Some of these features may be due to Cu binding to metallothioneins, but a larger proportion seems to result from biomineralization, most likely Cu(II) oxalate and Cu(II) oxides. Additional contributions in the EXAFS spectra indicate complexation of Cu(II) by the nonproteogenic amino acid nicotianamine, which has a very high affinity for Cu(II) as further characterized here.

Complexation and toxicity of copper in higher plants. I. Characterization of copper accumulation, speciation, and toxicity in Crassula helmsii as a new copper accumulator.

The amphibious water plant Crassula helmsii is an invasive copper (Cu)-tolerant neophyte in Europe. It now turned out to accumulate Cu up to more than 9,000 ppm in its shoots at 10 microm (=0.6 ppm) Cu(2+) in the nutrient solution, indicating that it is a Cu hyperaccumulator. We investigated uptake, binding environment, and toxicity of Cu in this plant under emerged and submerged conditions. Extended x-ray absorption fine structure measurements on frozen-hydrated samples revealed that Cu was bound almost exclusively by oxygen ligands, likely organic acids, and not any sulfur ligands. Despite significant differences in photosynthesis biochemistry and biophysics between emerged and submerged plants, no differences in Cu ligands were found. While measurements of tissue pH confirmed the diurnal acid cycle typical for Crassulacean acid metabolism, Delta(13)C measurements showed values typical for regular C3 photosynthesis. Cu-induced inhibition of photosynthesis mainly affected the photosystem II (PSII) reaction center, but with some unusual features. Most obviously, the degree of light saturation of electron transport increased during Cu stress, while maximal dark-adapted PSII quantum yield did not change and light-adapted quantum yield of PSII photochemistry decreased particularly in the first 50 s after onset of actinic irradiance. This combination of changes, which were strongest in submerged cultures, shows a decreasing number of functional reaction centers relative to the antenna in a system with high antenna connectivity. Nonphotochemical quenching, in contrast, was modified by Cu mainly in emerged cultures. Pigment concentrations in stressed plants strongly decreased, but no changes in their ratios occurred, indicating that cells either survived intact or died and bleached quickly.