Novel Bioassays Based On Functionalized Nanoparticles And Bionic Materials

Worldwide contamination of Cd as a consequence of industrial and agricultural activities is becoming a major environmental problem due to the great toxicity of Cd and its high mobility from soil to plants and further to the food chain. In China, presently, total area contaminated by heavy metals like Cd, As, Pb are approaching to2×107hm2, accounting for c.20%of the total cultivated area in our country. Particularly, the cultivated land contaminated by Cd is almost1.33×104hm2distributed in25districts in11provinces. Furthermore, Cd contamination is becoming heavier year by year. Therefore, the remediation and efficient use of Cd-contaminated soil in a safe way are of great importance for ecological restoration and sustainable development.Bechmeria nivea (L.) Gaud (ramie), indigenous to China, is an important fiber crop used for textile with a high tolerance to Cd stress as well as to drought and poor nutrition. It displays a high potential in the remediation of Cd-contaminated soil and water and ecological restoration of mine tailings. The main goal of this study was to investigate the accumulation of Cd and tolerance mechanisms in ramie. Furthermore, based on hydroponic and soil culture, we aimed to find out the role of this species in the efficient use of Cd-contaminated agriculture soils and re-vegetation of mine tailings. The main content and conclusions are as follows:In hydroponics, Cd accumulation in ramie was increased with external Cd concentrations. The highest Cd level occurred in root followed by shoot and the leaves had the least Cd concentrations. With Cd concentration in uptake media ranging from0.1to10mg L-1, Cd concentrations in root were4-60times higher than the above ground part where Cd concentrations were lower than100mg kg-1dw, indicating that ramie is not a Cd-hyperaccumulator. Cd translocation factor in ramie was decreased from0.29to0.04with external Cd increasing from0.1to10mg L-suggesting that tight control of Cd in sensitive tissues may act as an important defense line to counteract Cd toxicity in ramie. Cd uptake by the root of ramie followed Michaelis-Menten kinetics, with Km value of5.4mg L-1and Vmax value of99.3mg kg-1dw h-1, indicating that Cd uptake was a transporter-mediated process and gradually saturated. The accumulation of macro-and micro-nutrients by ramie were apparently affected by Cd stress:the level of Mg and Ca were significantly reduced by Cd in leaves. Low Cd (1mg L-1) stimulated the uptake of S, P and K, while with higher Cd (10mg L-1), the accumulation of S, P and K in leaves were markedly decreased. The levels of B、Mn、Fe、Cu in leaves were decreased at higher external Cd with Zn kept unaffected. In shoots, the concentrations of Mg and Ca were higher than the controls at1mg L-1Cd, while P and K were gradually decreased in a Cd concentration-dependent manner. This held true for the miicronutrients in shoot except B which kept stable at varying Cd concentrations. In roots, almost all the macro-and micro-nutrients uptakes were significantly increased at1mg L-1Cd except Ca which was reduced upon Cd exposure. At10mg L-1Cd, the increasing degree of each element was smaller except Ca. The permeability of cell membrane in ramie changed little at lower Cd concentration. While the maximum cell membrane permeability up to1.6times higher than the control was observed at10mg L-1Cd. Fumarate in leaves and roots as well as citric and malic acid in roots decreased with Cd in a concentration-dependent manner, suggesting that they were not likely to be involved in Cd detoxification mechanisms in ramie under Cd stress. Cellular accumulation of proline and spermidine were rapidly induced by Cd. As an effective buffer for osmotic stress, the increase of proline indicated the disturbance of water balance by Cd. As a quick response to Cd stress, the accumualtion of spermidine in ramie reflected its role in Cd tolerance.Determination of Cd distribution and chemical speciation in ramie is essential for understanding the mechanisms involved in Cd accumulation, transportation and detoxification. To assess the effect of Cd uptake on plant performance, nitrate reductase activity in leaves androot activity were analyzed during the entire experimental period. Subcellular fractionation of Cd-containing tissues indicated that about48.2-61.9%of the element was localized in cell walls and30.2-38.1%in soluble fraction, and the lowest in cellular organelles. Cd taken up by ramie rapidly equilibrated among different chemical forms. Results showed that the greatest amount of Cd was found in the extraction of1M NaCl and2%HAC, and the least in residues in all test tissues, suggesting that the majority of Cd was detoxified by integrating with pectates and protein followed by forming Cd-phosphate complexes in cells. In roots, the subdominant amount of Cd was extracted by d-H2O and80%ethanol, followed by0.6M HCl. While in stems and leaves, the amount of0.6M HCl-extractable Cd was comparable with that extracted by80%ethanol or d-F2O.1mg L-1Cd stimulated nitrate reductase activity in leaves and root activity, while a concentration-dependent inhibitory effect was observed with increasing Cd concentration, particularly at7mg L-1Cd. It could be suggested that the protective mechanisms evolved by ramie play an important role in Cd detoxification at relatively low Cd concentrations (below3mg L-1Cd) but become restricted to maintain internal homeostasis with higher Cd stress.Among the antioxidant mechanisms the ascorbate-glutathione cycle has been shown to play a central role in removing reactive oxygen species (ROS) and maintaining the cellular redox status.Ramie under Cd stress exhibited increased level of lipid peroxidation, as was evidenced by the increased malondialdehyde content (MDA, an index of lipid peroxidation) in leaves and roots.Shorter exposure to lower Cd concentrations (1and3mg L-’) led to a stimulation of chlorophyll synthesis, while longer exposure and higher Cd concentration (7mg L-1) led to a remarkable breakdown of chlorophyll, suggesting that Cd caused oxidative stress in ramie. The antioxidant system as represented by the ascorbate-glutathione pathway was clearly activated following Cd exposure. An overall increase in the metabolite levels and major constituting enzyme activities (ascorbate peroxidase APX and glutathione reductase GR) was observed in ramie treated with1and3mg L-1Cd throughout the entir experimental period, while prolongation of exposure to7mg L-1Cd resulted in decrease of GR activity and GSH pool in roots which may limit the operation of the whole cycle. Results suggest that Cd-induced oxidative damage in ramie is closely associated with the efficiency of its intrinsic antioxidant mechanisms and the accelerated operation of ascorbate-glutathione cycle provides ramie with enhanced Cd-stress tolerance.By hydroponics, it is understood that the threshold of ramie seedlings are7mg L-1, which is well above the concentrations of Cd (0.001-0.01mg L-1) exerting toxic effect to most organisms in aquatic system. It is concluded that ramie is a remarkable Cd-tolerant species. To further investigate the physiological response and tolerant mechanism of ramie to Cd in soil system, a series of soil Cd concentrations were set up according the typical Cd contamination degree in agriculture soils and mine tailings in Hunan province. As a result, Cd distribution pattern in ramie followed roots>shoots> leaves and the translocation factor was0.3, consistent with the results from the hydroponics. Ramie seedling in vegetative growth phase can tolerant up to80mg kg-1in soils. Within this range, the chlorophyll, carotenoid and soluble protein were all stimulated, and four key indices involving in AsA-GSH cycle including ascorbic acid (AsA、glutathione (GSH)、ascorbate peroxidation (APX)、glutathione reductase (GR) were all enhanced, which indicates that reactive oxygen species induced by Cd could be eliminated as much as possible by this acceleratory cycle. While the defending system of ramie suffered at higher Cd concentrations. It was proved that the optimum exogenous spm and Se4+concentrations for ramie was0.5×10-4mol L-1and1.26×10-6mol L-1, which could promote the content of chlorophyll, carotenoid and soluble protein significantly and inhibit the cell membrane-lipid peroxidation effectively. At the same time, the content of GSH, AsA and the activity of APX and GR were also increased markedly with exogenous spm. As a result, the whole cycle could operate at higher speed and this means a lot to strengthen the resistance of ramie to Cd.By analyzing the physiochemical characteristics and heavy metal concentrations in the agriculture soils around Qibaoshan mine and Mn tailings from Xiangtan Mn mine, it was found that elevated Cd, Cu and Zn concentration were approaching to or exceeded Chinese Environmental Quality Standards for soils. Particularly, Cd concentrations were up to8-fold higher than the third grade permitted by the soil standard. The Mn tailings contained rich N, P and K, which would benefit the survival of plants during re-vegetation. Total Mn (31903mg kg-1), Cd (119mg kg-1), Cu (126mg kg-1) and Zn (2490mg kg-1) in Mn tailings were all at phytotoxic levels, but did not differ significantly from those in rhizosphere soils. Mn tailings were clay textured, while rhizosphere soils were silty loam or clay loam. The compaction and anoxic nature of Mn tailings were considered to be the major constraints for plant establishment. By mixing the tailings with sand at1:1ratio, we tried to improve the aeration of the tailings, making it suitable for plant growth. The survival rate of ramie seedlings in the contaminated soils and improved tailings were100%and the growth of ramie greatly increased the bacteria amount in the rhizosphere. As compared to the control plants, the aboveground biomass of ramies grown in the tailings was reduced by30%while those grown in contaminated soils were unaffected. Presently, large scale of cultivated lands has been polluted by relatively low Cd concentrations. As a non-food crop, ramie is recommended to be cultivated in Cd-polluted land before food crops and vegetables are grown, which is considered as an efficient and safe way to use these types of soils. With low Cd concentration in leaves, ramie exhibits great potential for Cd phytostabilization rather than phytoextraction. Taking into account its rich root system, ramies can serves as good candidates for re-vegetation of degraded mining aera and soil and water conservation.