Although Cu is an essential component of several enzymes and other compounds in chloroplasts and mitochondria,it can be toxic at higher concentrations.Lastly, although I predicted that P would be correlated with metal content in tissues due to physicochemical sorption of phosphate to the ENMs, it was only in root tissue of HL plants exposed to CeO2 ENMs that a relationship was found. At root Ce concentrations below 100 µg g-1 , P was positively associated with Ce , but this trend plateaued at higher concentrations. One possible explanation for this is that CeO2 ENMs adsorbed P from the soil and were then sorbed into/onto the plant roots, but at higher exposure concentrations the soil was depleted of readily available P for the ENMs to adsorb. Previous studies using hydroponic systems have shown increased P uptake in maize exposed to ZnO ENMs and in spinach exposed to nZVI,although these results were due to the uptake of dissolved metal/phosphate complexes rather than ENM-sorbed P. Rui, et al.  observed the partial transformation of CeO2 ENMs into particulate CePO4 that were then taken up into hydroponically-grown cucumber seedlings, although the general lack of correlation between tissue Ce and P concentrations suggests this process was not occurring to a significant extent in this study.The issue metal concentrations of C. unguiculata grown in farm and grass soil were found to vary with ENM, soil, light level, concentration, and tissue type . While some of the general trends seen in potting soil were also seen in natural soils ,pot raspberries there was less total uptake in natural soils than in potting soil. In particular, ENMs in leaves were either present at very low concentrations or completely absent , which resulted in fewer impacts on plant physiology .

All three ENMs used here have been shown to have greatly reduced mobility in farm and grass soil compared to potting soil , which, in conjunction with an increase in aggregate size, is likely the cause of the decreased uptake seen here. CeO2 uptake was found to be limited almost exclusively to root tissue under all conditions, suggesting that only a very small fraction of CeO2 aggregates were transported through the roots to the vascular tissue and upwards to the stems and leaves. Despite their compositional similarities TiO2 was taken up in both roots and stems at much higher concentrations than CeO2, possibly as a result of their different behaviors in grass and farm soil, or because background Ti concentrations in the soil were two orders of magnitude higher than background Ce concentrations. As discussed in Chapter 2, CeO2 mean aggregate diameters in grass and farm soils and soil solutions were larger than those of TiO2 aggregates. Size exclusion has been implicated as one of the primary factors controlling plant uptake of ENMs and may be at least partially responsible for the results seen here. Both farm soil and grass soil have roughly an order of magnitude less available phosphate than potting soil , which appears to have allowed for ENMs to influence P uptake by C. unguiculata grown in natural soils. In grass soil, root P concentrations were positively correlated with both Ce and Cu concentrations for individuals exposed to those ENMs, although this effect was only seen under low light conditions. Since neither of these ENMs increase P mobility or bio-availability in grass soil , this correlation may be due to sorption of phosphate from the soil by the ENMs, which are then taken up into the roots. This is similar to what was seen in unfertilized potting soil, and provides further evidence that that under nutrient limited conditions metal oxide ENMs, particularly CeO2, are able to influence P bio-availability.ENM uptake and translocation in crop plants was found to vary with both species and illumination intensity . In contrast to C. unguiculata, however, increased ENM accumulation was seen at low rather than high light intensities. This is especially evident in wheat, and is most likely due to decreases in transpiration rates of both wheat and radishes grown in high light later in their life cycle .

Additionally, some ENM accumulation was found in the edible radish hypocotyls and wheat grains, particularly Ti in radish hypocotyls. Similar to C. unguiculata grown in farm soil, radishes accumulated very little to no metals in their leaves. However, radishes also had relatively low levels of ENMs in hypocotyl and root tissue although more root uptake occurred with CeO2 and TiO2 than Cu2 under low light conditions, possibly as a consequence of ENM surface charge. Zhu, et al. found increased uptake of positively charged Au ENMs over neutral or negatively charged particles in radish seedling root tissue grown hydroponically, so similar phenomena may be occurring here with the positively charged CeO2 and TiO2 and negatively charged Cu2. The low overall uptake of ENMs may also be related to the short lifespan of radishes, which is roughly half of the other species tested here. High concentrations of all three ENMs were found in wheat, particularly in individuals grown under low light conditions. On average, wheat had the highest concentrations of all three ENMs in all comparable tissues for all plant species, including C. unguiculata grown in potting soil. This last point is noteworthy for two reasons, namely, that all three ENMs have greatly decreased mobility in grass soil compared to potting soil and so the fraction of ENMs available for uptake is much smaller in grass soil, and that ENM uptake in wheat is known to be subject to strict size limitations. Larue, et al. 3 found that TiO2 ENMs above 140 nm did not accumulate in root tissues, those above 36 nm did not enter the cortex, but that ENMs 14 nm in diameter were able to pass through the CS, enter the vascular tissue, and be translocated throughout the plant. This aligns well with the distribution trends seen here and also shows that wheat plants are adept at taking up ENMs, even from soils where ENM mobility is highly limited. The presence of Cu in all tissues at concentrations significantly higher than the background provides further evidence that Cu2 undergoes at least partial dissolution in soil, especially when its uptake and translocation patterns in these plants are compared to those of the relatively insoluble TiO2 or CeO2.

Given that the Cu2 ENM used in these studies is a commercial biocide designed to release Cu ions, it is not surprising that this is the case. However, Chapter 5 shows that this ENM has harmful effects on the plants it is meant to protect, as Cu ions can be toxic to many plants at high concentrations. Since Cu2 is highly retained immediately at its point of entry to soil it may reach toxic concentrations in the soil even following application methods recommended by the manufacturer. However, while no significant correlations between Ce, Cu, or Ti content and P content were found in radishes, P and Cu concentrations had a significant positive relationship in the leaves and stems of wheat grown under low light conditions. This suggests relatively high soil concentrations of Cu2 may be able to enhance P uptake by certain plants.Copper is toxic to life at levels that vary depending on the organism. Humans are mandated to not exceed 1–2 mg/L copper in their drinking water ,plastic gardening pots while some freshwater animals and plants experience acute toxic effects at concentrations as low as 10 µg/L . Because the human food chain begins with plants, it is critical to understand how plants tolerate heavy metals including copper, which is frequently concentrated in soils as a result of pesticide application, sewage sludge deposition, mining, smeltering, and industrial activities. This issue is also at the crux of applying phytoremediation approaches, which use green plants to decontaminate or contain polluted soils and sediments and to purify waste waters and landfill leachates . Metal-tolerant plants inhibit incorporation of excess metal into photosynthetic tissue by restricting transport across the root endodermis and by storage in the root cortex . In contrast, hyper accumulating plants extract metals from soils and concentrate excess amounts in harvestable parts such as leaves. Copper detoxification seems to be linked to mechanisms that bind Cu to molecular thiol groups. Cysteine-rich peptides, such as phytochelatins which transport copper to the shoot, increase in response to high cellular levels of Cu , and Cu-S binding occurs in roots and leaves of Larrea tridentata. However, an unidentified copper species, concentrated in electron-dense granules on cell walls and some vacuole membranes, appears to be the main morphological form of copper sequestered in Oryza sativa , Cannabis sativa , Allium sativum , and Astragalus sinicus . Plants take in and exclude elements largely at the soil-root interface within the rhizosphere, i.e. the volume of soil influenced by roots, mycorrhizal fungi, and bacterial communities . Deciphering processes that control the bio-availability of metals in the field is difficult because the rhizosphere is compositionally and structurally complex. Here we report on using synchrotron-based microanalytical and imaging tools to resolve processes by which metaltolerant plants defend themselves against excess cationic copper.

We have mapped the distribution of copper in self-standing thin sections of unperturbed soils using micro-Xray fluorescence and identified structural forms of copper at points-of-interest using micro-extended X-ray absorption fine structure spectroscopy and X-ray diffraction . Because only a few small areas could be analyzed in reasonable times with micro-analyses, the uniqueness of the micro-analytical results was tested by recording the bulk EXAFS spectrum from a sample representing the entire rhizosphere and by simulating this spectrum by linear combination of copper species spectra from POIs. We investigated copper speciation in rhizospheres of Phragmites australisand Iris pseudoacorus, two widespread wetland species with high tolerances to heavy metals .P. australisis frequently used to treat waste waters because it can store heavy metals as weakly soluble or insoluble forms. Its roots can be enriched in Cu 5-60 times relative to leaves, with large differences among ecotypes and between field-grown versus hydroponically grown plants . To take into account natural complexity, including any influence of bacteria, fungi, or climate variation, our experiment was conducted outdoors, rather than in a greenhouse on seedlings using ex-solum pots or hydroponic growth methods. The soil was from the Pierrelaye plain, a 1200 ha truck-farming area about 30 km northwest of Paris, France. From 1899 to 1999, regular irrigation of the Pierrelaye plain with untreated sewage water from Paris caused contamination with heavy metals, mainly Zn, Pb, and Cu . Such pollution is pervasive worldwide because increasing populations and associated economic growth are diminishing available freshwater, thus leading to increased irrigation of farmlands with waste waters.In the initial soil, copper occurs in two morphological forms . One form decorates coarse organic particles that have some recognizable structures from reticular tissue , and the other occurs inthe fine clayey matrix in areas that show organic particulate shapes only at high µ-XRF resolution. In the two phytore mediated soils, similar Cu-organic particulate associations, but also, hot spots of Cu grains 5-20 µm in size were observed in the thin-section maps . In the rhizosphere of P. australis, the Cu hot spots exist outside and in roots and specifically in cortical parenchyma, but not in central vascular cylinders from the stele that contain vascular bundles through which micro-nutrients are transported to reproductive and photosynthetic tissues. In contrast, the main roots and rhizome of I. pseudoacorus do not contain detectable Cu grains, but in the surrounding soil Cu grains are aligned, suggesting that they are associated with biological structures. Under an optical microscope filamentous and ramified organic structures, similar to root hairs or hyphae from endomycorrhizal fungi, are visible in places where the Cu spots were observed by µ-XRF . Fungal forms are more likely because mycorrhizal hyphae typically are anastomosing, whereas root hairs are not. Fungi may also be implicated in the formation of Cu grains in the cortex of P. australis since roots of this plant are known to be colonized by arbuscular endomycorrhizae in contaminated environments . These hypotheses are consistent with the capacity of mycorrhizae to accumulate metals and with the storage of Cu in secondary feeder roots of the water hyacinth Eichhornia crassipes .