The Cu grains have about the same size as the electron-dense Cu granules in cells of E. splendens placed in a CuSO4 solution for 30 days .While biological activity clearly modified the original distribution of Cu in the rhizospheres, the Cu species could not be identified from the µ-XRF maps but instead were elucidated using EXAFS spectroscopy. All eight µ-EXAFS spectra from areas in the original soil containing the particle morphologies and chemical compositions observed with µ-XRF can be superimposed on the soil’s bulk EXAFS spectrum , indicating that the initial Cu speciation occurred uniformly. If the initial soil contained various assemblages of Cu species that were distributed unevenly, then we might expect that the proportions of species also would have varied among analyzed areas and been detectable by µ-EXAFS spectroscopy ; however, this was not observed. These spectra match those for Cu2+ binding to carboxyl ligands in natural organic matter, as commonly observed . Elemental Cu. In contrast, only the reference spectrum of elemental copper matches the µ-EXAFS spectra of the 12 hot-spot Cu grains, which are statistically invariant . Photo reduction of Cu2+ in the X-ray beam cannot explain the formation of Cu0 because no elemental Cu was detected in the initial soil by powder and µ-EXAFS, and Cu0 was detected in the two phytoremediated soils at 10 K by bulk EXAFS, and all individual spectral scans from the same sample could be superimposed. At 10 K, radiation damage is delayed , and if Cu had been reduced in the beam, the proportion of Cu0 would have increased from scan to scan which was not observed.The rhizospheres were oxidizing as indicated by the presence of iron oxyhydroxide , absence of sulfide minerals, and the fact that P. australis and I. pseudoacorus are typical wetlands plants with aerenchyma that facilitate oxygen flow from leaves to roots . Thermodynamic calculations using compositions of soil solutions collected below the rhizosphere indicate that Cu+ and Cu2+ species should have been dominant . These points along with the occurrences of nanocrystalline Cu0 in plant cortical cells and as stringer morphologies outside the roots together suggest that copper was reduced biotically. Ecosystem ecology of the rhizosphere indicates synergistic or multiple reactions by three types of organisms: plants, endomycorrhizal fungi, and bacteria.

Normally, organisms maintain copper homeostasis through cation binding to bio-active molecules such as proteins and peptides. When bound,plastic pots for planting the Cu2+/Cu1+ redox couple has elevated half-cell potentials that facilitate reactions in the electron-transport chain. Even though average healthy cell environments are sufficiently reducing , there are enough binding sites to maintain copper in its two oxidized states. Copper is also important in controlling cell-damaging free radicals produced at the end of the electron-transport chain, for example in the superoxide dismutase enzyme Cu-Zn-SOD, which accelerates the disproportionation of superoxide to O2 and hydrogen peroxide. However, unbound copper ions can catalyze the decomposition of hydrogen peroxide to water and more free radical species. To combat toxic copper and free radicals, many organisms overproduce enzymes such as catalase, chelates such as glutathione, and antioxidants . Mineralizationcould also be a defense against toxic copper, but reports of Cu+ and Cu2+ biominerals are rare; only copper sulfide in yeast and copper oxalate in lichens and fungi are known. Atacamite 3Cl in worms does not appear to result from a biochemical defense. Biomineralization of copper metal may have occurred by a mechanism analogous to processes for metallic nanoparticle synthesis that exploit ligand properties of organic molecules. In these processes, organic molecules are used as templates to control the shape and size of metallic nanoparticles formed by adding strong reductants to bound cations. For copper nanoparticles and nanowires, a milder reductants ascorbic acids has been used. Ascorbic acid, a well-known antioxidant, reduces Cu cations to Cu0 only when the cations are bound to organic substrates such as DNA in the presence of oxygen in the dark or via autocatalysis on Cu metal seeds in the absence of stabilizing organic ligands . As an example of synthetic control, pH dependent conformation of histidine-rich peptides has led to larger nanocrystals of Cu0 at pH 7–10 than at pH 4–6 . Plants produce ascorbic acid for many functions and rhizospheres often contain the breakdown products of ascorbic acid, which facilitates electron transfer during mineral weathering . Plants produce more ascorbic acid when grown in soils contaminated with heavy metals including copper . Fungi, which proliferate over plants and bacteria in metal-contaminated soils, can stabilize excess copper by extracellular cation binding or oxalate precipitation , but mechanisms probably also require enzymes, thiol-rich proteins and peptides, and antioxidants .

The formation of electron-dense Cu granules within hyphae of arbuscular mycorrhizal fungi isolated from Cu- and As-contaminated soil suggests that fungi also can produce nanoparticulate copper. Some copper reduction possibly occurred in response to the European heat wave of the summer of 2003 . Elevated expression of heat shock protein HSP90 and metallothionein genes has been observed in hyphae of an arbuscular mycorrhizal fungus in the presence of 2 × 10–5 M CuSO4 in the laboratory . This suggests that a single driving force can trigger a biological defense mechanism that has multiple purposes. Thus, reduction of toxic cations to native elements may increase as rhizosphere biota fight metal stress and stresses imposed by elevated temperatures expected from global warming.Laboratory evidence has shown that plants , fungi , bacteria , and algae can transform other more easily reducible metals, including Au, Ag, Se , Hg, and Te, to their elemental states both intra and extracellularly. When mechanisms have been proposed, they typically have involved enzymes; however, ascorbic acid was implicated when Hg2+ was transformed to Hg0 in barley leaves . Theoretically all of these metal cations could be transformed by a reducing agent weaker than ascorbic acid . However, binding appears to stabilize cationic forms in the absence of a sufficiently strong reductant such as ascorbic acid. Processes used in materials synthesis that were developed with biochemical knowledge might yield clues to other possible, but presently unknown, biologically mediated reactions in different organisms.The discovery of nanoparticulate copper metal in phytoremediated soil may shed light on the occurrence of copper in peats. Native copper likely forms abiotically in the reducing acidic environments of Cu-rich peat bogs . However, swamps by definition are more oxidizing with neutral to alkaline pHs, and they may be ideal sites for biotic formation of metallic Cu nanoparticles. For example, in swamp peats near Sackville, New Brunswick, Canada, copper species unidentifiable by XRD were dissolved only with corrosive perchloric acid , suggesting they may have been nanoparticulate metal formed by active root systems of swamp plants. If swamp peats evolve to bog peats the Cu reduction mechanism could convert to autocatalysis on the initial nanocrystals . The addition of peats that either act as templating substrates or contain nanoparticulate copper could enhance the effectiveness of using wetlands plants for phytoremediation. In contrast to harvesting hyperaccumulators, the oxygenated rhizosphere would become an economic source of biorecycled copper, and rhizosphere containment would prevent copper from entering the food chain via herbivores,plant pot drainage limiting potential risks to humans.Predicting Cd concentrations in plants is essential for controlling Cd entry into the food chain.

Cadmium uptake by plants is the result of root adsorption to cell walls and of absorption through root cell membranes. Concentration-dependent kinetics performed on seedlings of maize and alpine pennycress allowed short-term Cd uptake by both root uptake pathways to be parameterized . However, the uptake parameters were obtained on plants which were previously cultivated without exposure to Cd. The objective of the present work was to assess how chronic exposure of plants to different Cd levels would affect the root adsorption and absorption rates. Indeed, pre-exposure to metals may substantially modify the kinetics of metal uptake. Stimulation of Cd uptake has been reported to occur in Fe deficient conditions , showing some up-regulation of membrane proteins able to transport Cd. Moreover, Larsson et al. studied the effect of prior Cd2+ exposure on Cd uptake by roots of Arabidopsis thaliana, and found some up-regulation of total Cd uptake in the wild type, and down-regulation in the PC-deficient mutant. Furthermore, very few works have investigated the regulation of root cation exchange capacity by prior exposure to metal. In the present study, we investigated the impact of plant Cd content on root Cd uptake characteristics, at both the cell wall and membrane levels. The experiment was performed on two species with contrasting demand for Cd: a hyper accumulating ecotype of alpine pennycress , well-known for its ability to accumulate high concentrations of Zn and Cd in its shoots, and maize , which retains Cd in its roots. The remaining plants were exposed to a one-hour uptake in a radio-labelled solution in order to assess the absorption rate of Cd according to the level of Cd accumulated during the cultivation. Three Cd concentrations in the radio-labelled solution were used: 0.1 µM, 10 µM and 50 µM. Before immersion of roots in the radio-labelled solution, they were rinsed and exposed to a desorption treatment in order both to minimize contamination of the radio-labelled solution with Cd leakage from the cell walls, and to liberate all exchange sites able to adsorb Cd. For that, each root system was immersed for two hours in 80 ml of buffered solution containing 5 mM of Ca2 and 2 mM MES buffer, then for two further hours in 80 ml of buffered solution containing 0.5 mM of Ca2 and 2 mM MES buffer. After quick rinsing in distilled water, roots were immersed in 650 ml of 109Cd radio-labelled solution containing 0.5 mM CaCl2, 2 mM MES buffer and CdCl2 in the three different concentrations. Root exposure lasted one hour, without significant variation of Cd external concentration. Each root system was then separated from shoots before immersion in successive ice-cold MES-buffered baths containing 2 mM CdCl2 and 5 mM CaCl2. This desorption procedure was interrupted by 2 minutes freezing in liquid nitrogen; roots were then thawed by agitation in a warm desorbing bath, and desorption kinetics went on in ice-cold desorbing solutions for the resulting disrupted root cells . Cadmium collected in the desorbing solutions was quantified through 20 ml samples by gamma-counting .

Cadmium bound to the apoplast at the end of desorption was also quantified through gamma-counting in dry matter. We previously showed that the sudden unloading of metal in the desorption solution caused by the freezing/thawing procedure represents symplastic Cd, while the gradual desorption before and afterwards, corresponds to leakage from the cell walls, regardless of the external concentration . The HATS of maize and alpine pennycress is not affected at all by the low internal accumulation of Cd during growth. The withdrawal of free Cd ion from the cytosol by the complexation with phytochelatins and eventual transport to the vacuole or to the shoots may depress any mechanism regulating the cytosolic Cd concentrations . On the other hand, the plants do not show the same behaviour for the high short-term exposure concentrations: the 0.1 µM Cd contamination of the growth solution would down-regulate the LATS of maize but not that of alpine penny cress, for which some stimulating effect seems to happen. After high Cd accumulation during growth, the Cd symplastic influx is significantly reduced for both plants. Such a diminution of the intracellular uptake of Cd had already been observed on wheat root protoplasts . It may come from down-regulation of the short-time Cd2+ uptake on both high- and low-affinity transport systems. In our study, this reduction of the Cd short-term uptake is higher for the lower exposure concentrations. This supposes that the down-regulation affects the HATS rather than the LATS. The decrease in Cd intracellular influx can also result from up-regulation of the Cd2+ extrusion from intracellular to extracellular space, through PC-Cd efflux , Cd2+/H+ antiport or vesicle excretion . Another alternative explanation to the lower Cd uptake could be the changed appearance of the root system in the Cd-treated plants. Thus, although approximately the same root masses were achieved in all plants tested, the uptake surface might have been different, favoring Cd uptake in the control plants. Finally, some better sequestration of Cd by the cell walls might decrease the entry rate of Cd through cell membranes.