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).

Ultratrace Metal Speciation Analysis by Coupling of Sector-Field ICP-MS to High-Resolution Size Exclusion and Reversed-Phase Liquid Chromatography.

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.

Sexual Dimorphism in the Response of Mercurialis annua to Stress.

The research presented stemmed from the observations that female plants of the annual dioecious Mercurialis annua outlive male plants. This led to the hypothesis that female plants of M. annua would be more tolerant to stress than male plants. This hypothesis was addressed in a comprehensive way, by comparing morphological, biochemical and metabolomics changes in female and male plants during their development and under salinity. There were practically no differences between the genders in vegetative development and physiological parameters. However, under salinity conditions, female plants produced significantly more new reproductive nodes. Gender-linked differences in peroxidase (POD) and glutathione transferases (GSTs) were involved in anti-oxidation, detoxification and developmental processes in M. annua. ¹H NMR metabolite profiling of female and male M. annua plants showed that under salinity the activity of the TCA cycle increased. There was also an increase in betaine in both genders, which may be explainable by its osmo-compatible function under salinity. The concentration of ten metabolites changed in both genders, while 'Female-only-response' to salinity was detected for five metabolites. In conclusion, dimorphic responses of M. annua plant genders to stress may be attributed to female plants' capacity to survive and complete the reproductive life cycle.

Selenium species in the roots and shoots of chickpea plants treated with different concentrations of sodium selenite.

The trace element selenium has an essential role for human health. It is involved in redox center functions, and it is related to the immune system response. Legumes are among the main suppliers of selenium into the human food chain. Not only Se concentration as such but also more the chemical species of Se is of higher importance for successful Se supply to the human diet and its bioavailability. The current study was focused on the investigation of the Se species present in chickpea plants exposed to 0, 10, 25, 50, and 100 µM selenite in short- and long-term treatment studies. The linear increase of total Se concentration could be linked to the increased concentrations of Se exposure. The selenium species (SeMet, SeCys, selenite, selenate, GPx) detected in varying concentrations in shoots and roots depend on the exposure's concentration and duration. The investigation showed that chickpea can accumulate Se in favorable concentrations and its transformation to bioavailable Se species may have positive impacts on human health and aid to implement Se into the diet.

Comparative study on the impact of copper sulphate and copper nitrate on the detoxification mechanisms in Typha latifolia.

The present study focused on cupric sulphate and cupric nitrate uptake in Typha latifolia and the impact of these copper species on the plant's detoxification capacity. When the plants were exposed to 10, 50 and 100 µM cupric sulphate or cupric nitrate, copper accumulation in T. latifolia roots and shoots increased with rising concentration of the salts. Shoot to root ratios differed significantly depending on the form of copper supplementation, e.g. if it was added as cupric (II) sulphate or cupric (II) nitrate. After incubation with 100 µM of cupric sulphate, up to 450 mg Cu/kg fresh weight (FW) was accumulated, whereas the same concentration of cupric nitrate resulted in accumulation of 580 mg/kg FW. Furthermore, significant differences in the activity of some antioxidative enzymes in Typha roots compared to the shoots, which are essential in the plant's reaction to cope with metal stress, were observed. The activity of peroxidase (POX) in roots was increased at intermediate concentrations (10 and 50 µM) of CuSO4, whereas it was inhibited at the same Cu(NO3)2 concentrations. Ascorbate peroxidase (APOX) and dehydroascorbate reductase (DHAR) increased their enzyme activity intensely, which may be an indication for copper toxicity in T. latifolia plants. Besides, fluorodifen conjugation by glutathione S-transferases (GSTs) was increased up to sixfold, especially in roots.

The fate of arsenic, cadmium and lead in Typha latifolia: a case study on the applicability of micro-PIXE in plant ionomics.

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.

Localization and quantification of Pb and nutrients in Typha latifolia by micro-PIXE.

Typha latifolia is a plant species widely used for phytoremediation. Accumulation, localization and distribution of Pb and mineral nutrients were investigated in roots, rhizomes and leaves of Typha latifolia grown at 0, 50, 100 and 250 µM Pb concentrations in a pot experiment under controlled conditions. Bulk elemental concentrations were determined by X-ray fluorescence (XRF) spectroscopy whereas micro-proton-induced X-ray emission (micro-PIXE) was used for element localization in root and rhizome tissues. Gradual increase in bulk Pb concentrations was observed in Typha latifolia roots and rhizomes treated with increasing Pb concentrations, however in rhizomes Pb concentrations were an order of magnitude lower than in roots. In leaves Pb concentrations were around the limit of detection for XRF (~20 µg g(-1)). An increase in concentration of K and Ca in roots, rhizomes and leaves, of iron and zinc in roots and leaves, and of Mn in rhizomes was observed either at 50 and/or 100 µM Pb treatments, whereas for K and Ca in roots, rhizomes and leaves, Fe and Zn in roots and leaves and Mn in rhizomes, or at 250 µM Pb treatment the increase was seen for concentrations of Fe and Zn in rhizomes and Cu in roots. Mn concentrations decreased with Pb treatments in roots and leaves. Element localization using micro-PIXE analysis demonstrated Pb accumulation in epidermal and cortical tissues of treated roots and rhizomes, while in endodermis and vascular tissues Pb was not detected. A displacement of Ca from epidermal to cortical tissues was observed in Pb treated roots and rhizomes, pointing to cell wall immobilization of Pb as one of the tolerance mechanisms in Typha latifolia. High level of colocalization of Pb with P (r = 0.60), S (r = 0.37) and Zn (r = 0.70) was observed in Pb treated roots, while in rhizomes colocalization with the mentioned elements was still positive, but not that prominent. These results indicate that Pb may form complexes with phosphorus and sulfur compounds in roots and rhizomes, which may also represent attraction sites for binding Zn. Because of its large root and rhizome surface area acting as main sites for Pb adsorption, Typha latifolia may represent potentially efficient plant species for phytoremediation of Pb contaminated soils and waters.

Plants for waste water treatment--effects of heavy metals on the detoxification system of Typha latifolia.

Upon treatment with Cd and As cattail (Typha latifolia) showed induced catalase, monodehydroascorbate reductase and ascorbate peroxidase activities in leaves but strong inhibition in rhizomes. Peroxidase activity in leaves of the same plants was inhibited whereas linear increase was detected after Cd treatment in rhizomes. Glutathione S-transferase measurements resulted in identical effects of the trace elements on the substrates CDNB, DCNB, NBC, NBoC, fluorodifen. When GST was assayed with the model substrate DCNB, a different pattern of activity was observed, with strongly increasing activities at increasing HM concentrations. Consequently, to improve the success rates, future phytoremediation plans need to preselect plant species with high antioxidative enzyme activities and an alert GST pattern capable of detoxifying an array of organic xenobiotics.

Response of antioxidant enzymes in Nicotiana tabacum clones during phytoextraction of heavy metals.

BACKGROUND, AIM, AND SCOPE: Tobacco, Nicotiana tabacum, is a widely used model plant for growth on heavy-metal-contaminated sites. Its high biomass and deep rooting system make it interesting for phytoextraction. In the present study, we investigated the antioxidative activities and glutathione-dependent enzymes of different tobacco clones optimized for better Cd and Zn accumulation in order to characterize their performance in the field. MAIN FEATURES: The improved heavy metal resistance also makes the investigated tobacco clones interesting for understanding the plant defense enzyme system in general. Freshly harvested plant material (N. tabacum leaves) was used to investigate the antioxidative cascade in plants grown on heavy metal contaminated sites with and without amendments of different ammonium nitrate and ammonium sulfate fertilizers. MATERIALS AND METHODS: Plants were grown on heavily polluted soils in north-east Switzerland. Leaves were harvested at the field site and directly deep frozen in liquid N(2). Studies were concentrated on the antioxidative enzymes of the Halliwell-Asada cycle, and spectrophotometric measurements of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), superoxide dismutase (SOD, EC 1.15.1.1), glutathione peroxidase (GPX, EC 1.11.1.9), glutathione reductase (GR, EC 1.6.4.2), glutathione S-transferase (GST, EC 2.5.1.18) were performed. RESULTS AND DISCUSSION: We tried to explain the relationship between fertilizer amendments and the activity of the enzymatic defense systems. When tobacco (N. tabacum) plants originating from different mutants were grown under field conditions with varying fertilizer application, the uptake of cadmium and zinc from soil increased with increasing biomass. Depending on Cd and Zn uptake, several antioxidant enzymes showed significantly different activities. Whereas SOD and CAT were usually elevated, several other enzymes, and isoforms of GST were strongly inhibited. CONCLUSIONS: Heavy metal uptake represents severe stress to plants, and specific antioxidative enzymes are induced at the cost of more general reactions of the Halliwell-Asada cycle. In well-supplied plants, the glutathione level remains more or less unchanged. The lack of certain glutathione S-transferases upon exposure to heavy metals might be problematic in cases when organic pollutants coincide with heavy metal pollution. When planning phytoremediation of sites, mixed pollution scenarios have to be foreseen and plants should be selected according to both, their stress resistance and hyperaccumulative capacity.

Do heavy metals and metalloids influence the detoxification of organic xenobiotics in plants?

BACKGROUND, AIM AND SCOPE: Mixed pollution with trace elements and organic industrial compounds is characteristic for many spill areas and dumping sites. The danger for the environment and human health from such sites is large, and sustainable remediation strategies are urgently needed. Phytoremediation seems to be a cheap and environmentally sound option for the removal of unwanted compounds, and the hyperaccumulation of trace elements and toxic metals is seemingly independent from the metabolism of organic xenobiotics. However, stress reactions, ROS formation and depletion of antioxidants will also cause alterations in xenobiotic detoxification. Here, we investigate the capability of plants to detoxify chlorophenols via glutathione conjugation in a mixed pollution situation. MATERIALS AND METHODS: Typha latifolia and Phragmites australis plants for the present study were grown under greenhouse conditions in experimental ponds. A Picea abies L. suspension culture was grown in a growth chamber. Cadmium sulphate, sodium arsenate and lead chloride in concentrations from 10 to 500 microM were administered to plants. Enzymes of interest for the present study were: glutathione transferase (GST), glutathione reductase, ascorbate peroxidase and peroxidase. Measurements were performed according to published methods. GST spectrophotometric assays included the model substrates CDNB, DCNB, NBC, NBoC and the herbicide Fluorodifen. RESULTS: Heavy metals lead to visible stress symptoms in higher plants. Besides one long-term experiment of 72 days duration, the present study shows time and concentration-dependent plant alterations already after 24 and 72 h Cd incubation. P. abies spruce cell cultures react to CdSO(4) and Na(2)HAsO(4) with an oxidative burst, similar to that observed after pathogen attack or elicitor treatment. Cd application resulted in a reduction in GSH and GSSG contents. When a heavy metal mixture containing Na(2)HAsO(4), CdSO(4) and PbCl(2) was applied to cultures, both GSH and GSSG levels declined. Incubation with 80 microM arsenic alone doubled GSSG values. Based on these results, further experiments were performed in whole plants of cattail and reed, using cadmium in Phragmites and cadmium and arsenic in Typha as inducers of stress. In Phragmites australis, GST activities for CDNB and DCNB were significantly reduced after short-term Cd exposure (24 h). In the same samples, all antioxidant enzymes increased with rising heavy metal concentrations. Typha latifolia rhizome incubation with Cd and As leads to an increase in glutathione reductase and total peroxidase activity and to a decrease in ascorbate peroxidase activity. Measurements of the same enzymes in leaves of the same plants show increased GR activities, but no change in peroxidases. GST conjugation for CDNB was depressed in both cattail rhizomes and leaves treated with Cd. After As application increased, DCNB enzyme activities were detected. DISCUSSION: T. latifolia and P. australis are powerful species for phytoremediation because they penetrate a large volume of soil with their extensive root and rhizome systems. However, an effective remediation process will depend on active detoxifying enzymes, and also on the availability of conjugation partners, e.g. glutathione and its analogues. Species-specific differences seem to exist between the regulations of primary defence enzymes like SOD, catalase, peroxidases, whereas others prefer to induce the glutathione-dependent enzymes. As long as the pollutant mix encountered is simple and dominated by heavy metals, plant defence might be sufficient. When pollution plumes contain heavy metals and organic xenobiotics at the same time, this means that part of the detoxification capacity, at least of glutathione-conjugating reactions, is withdrawn from the heavy metal front to serve other purposes. In fact, glutathione S-transferases show strong reactions in stressed plants or in the presence of heavy metals. The spruce cell culture was a perfect model system to study short-term responses on heavy metal impact. Overall, and on the canopy level, this inhibitory effect might result in a lower detoxification capacity for organic pollutants and thus interfere with phytoremediation. CONCLUSIONS: We present evidence that pollution with heavy metals will interfere with both the oxidative stress defence in plants, and with their ability to conjugate organic xenobiotics. Despite plant-species-dependent differences, the general reactions seem to include oxidative stress and an induction of antioxidative enzymes. Several processes seem to depend on direct binding of heavy metals to enzyme proteins, but effects on transcription are also observed. Induction of xenobiotic metabolism will be obtained at high heavy metal concentrations, when plant stress is elevated. RECOMMENDATIONS AND PERSPECTIVES: Plants for phytoremediation of complex pollution mixtures have to be selected according to three major issues: uptake/accumulation capacity, antioxidative stress management, and detoxification/binding properties for both the trace elements and the organic xenobiotics. By way of this, it might be possible to speed up the desired remediation process and/or to obtain the desired end products. And, amongst the end products, emphasis should be laid on industrial building materials, biomass for insulation or biogas production, but not for feed and fodder. Each of these attempts would increase the chances for publicly accepted use of phytoremediation and help to cure the environment.

Short communication direct effect of Cd on glutathione S-transferase and glutathione reductase from Calystegia sepium.

Interactions between heavy metals, glutathione, glutathione S-transferase (GST), and glutathione reductase (GR) are being investigated by many working groups, but evaluation of the direct effect of Cd+ on these enzymes in vitro is lacking. We report here the effect of cadmium (10, 50, 100, 250 microM CdSO4) on partially purified enzymes from Calystegia sepium. Plants were grown under normal field conditions without metals and the enzymes were extracted by Tris buffer and partially purified by ammonium sulphate fractionation and gel filtration. Glutathione S-transferase activity was measured with different substrates, i.e., 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrobenzylchloride (NBC), and the herbicide Fluorodifen. GST activity was significantly lower in leaf compared to stem, flower, and rhizome and the inhibitory effect of Cd was obtained with NBC and Fluorodifen substrates at 250 microM. There was no effect of Cd on GR activity up to 250 microM.