| Copper (Cu) is an essential trace element for plant, excess Cu has a adverse impact on plant growth, photosynthesis and root activity, and through the food chain, directly or indirectly affects human health. Therefore, the clean up of heavy metal contaminated soils is a very urgent task. Chelate-induced phytoremediation and an environmental friendly technique in situ for cleaning up heavy metals from contaminated land have been paid more and more attention in recent years, which is a low cost and is able to maintain soil structure and fertility. Many applied researches on chelate-induced phytoremediation have been done, but there are few experimental studies on the mechanism. Many issues remain unclear about the mechanism.In this study, a natural, completely biodegradable aminomulticarboxyl chelating agents [S, S"]-ethylenediamine disuccinic acid (EDDS) and maize (Zea Mays L. Zhengda 958) were used for the experimental materials. Cu accumulation mechanism induced by EDDS and its effects on plant growth were studied by using both hydroponics and soil cultivation. The contents of Cu and Cu-EDDS complex in shoots and roots, xylem sap, different parts of primary roots and sub-cellular components such as protoplasts and vacuoles were analyzed respectively by using inductively coupled plasma atomic emission spectrometry (ICP-AES) and ion chromatography (IC). Absorption sites, pathways and patterns of Cu-EDDS were systematically and quantificationally studied by using ICP-AES, IC and rubeanic acid histochemical method. The major findings made from the above experiments were as following:Anew analysis method, suitable for the complexes of heavy metals (iron, Cu, Pb) and EDDS in plant and soil, was developed by using an anion-exchange column and an UV-visible light detector, and the method was confirmed by using the electrospray ionization mass spectrum. The detection limits of Fe (?)-, Pb-and Cu-EDDS complexes were 0.38,0.54 and 0.18 ?mol L-1, and the recoveries were 99.6%,100.6% and 97.5%, respectively. The method is simple, sensitive and suitable for analyzing alone or simultaneously Fe (?)-, Cu-and Pb-EDDS complexes in plant and soil.The EDDS treatment had significant dual effects on maize plant growth, photosynthesis and root activity. When EDDS concentration was lower than the free Cu2+ concentration in culture solution, maize plant biomass yield, net photosynthesis rate and root activity would increase with EDDS concentration increase, when more than the free Cu2+ concentration in the solution, the excessive EDDS could lead to decrease of plant biomass yield, photosynthesis rate and root activity. Compared with 100 ?mol L-1 Cu2+ treatment alone, adding 25-400 ?mol L-1 EDDS could significantly reduce the Cu contents in shoot and roots, and the contents could decrease with the EDDS concentration increase. EDDS treatments decreased Cu accumulation level in shoots and roots by formation Cu-EDDS complex to reduce the Cu2+ ions concentration (activity) in the solution. The absorption rate for Cu and Cu-EDDS complex in roots was a restrictive factor to determine Cu accumulation level in shoots.The treatments with 200-1200 ?mol L-1 Cu-EDDS complex could significantly increase the Cu contents of shoots and roots. The Cu content of roots (y) had a positive correlation with Cu-EDDS complex concentration (x), y= 0.3222x+27.674 (R2= 0.9783), and the Cu contents of shoots had an "S"-shaped curve relationship with the Cu-EDDS complex concentration. Cu-EDDS complex concentration of xylem sap significantly increased with the solution Cu-EDDS complex concentration increase, and the Cu content in shoots significantly increased with Cu-EDDS complex concentration in xylem sap. The results showed that there were two different Cu absorption systems in maize roots, one was an active absorption system for Cu2+ ions, and the other was a passive absorption system for Cu-EDDS complex. The decrease of membrane permeability, under the hydroponics condition, was the key factors limiting the absorption of Cu-EDDS complex.The results from the pot experiments showed that shoot Cu contents had very significant correlation with the soil soluble Cu contents, Cu accumulation levels in roots and cell membrane permeability, the correlation coefficients were 0.968,0.966 and 0.952. The chelated Cu (Cu-EDDS complex) in shoots, roots and xylem sap accounted for 63.91%-51.27%of total Cu in shoots,4.70%-3.82% of total Cu in roots and 86.93%-87.65% of total Cu in xylem sap, respectively. The root injuries could significantly improve the absorption and accumulation of Cu-EDDS complex, and compared with CK (600 ?mol L-1 Cu-EDDS complex alone). The Cu contents in shoots increased by 7.59 times,5.10 times and the Cu contents in roots increased by 13.44 times,9.00 times, respectively.The absorption sites, pathways and mechanism of Cu-EDDS complex in primary roots of maize were related to EDDS concentrations. Under physiological and lower concentrations of Cu-EDDS complex treatment conditions, the complex was absorbed by diffusing through the apoplastic places where lateral roots occurred and the apical parts without endodermis Casparian strips into the root xylem. Besides these sites, under the high concentrations (>3000?mol L-1) Cu-EDDS complex hydroponics and soil culture conditions, the complex might result in injury of the physiological barrier of controlling ion absorption, therefore the complex might enter the root xylem from the passage cells and adjacent early metaxylem vessels. Lateral root zones were the main absorption sites of Cu-EDDS complex, the apoplastic bypass was its main pathway entering the root xylem and the Casparian strips were its main physical barriers.The treatment with 2-4 mmol EDDS Kg-1 (soil) had an important impact on the subcellular distribution of Cu and Cu-EDDS complex in leaf and root cells. Compared with the control, the major parts of Cu in leaf and root cells were still present in the cell walls, but the proportions of the Cu contents in cytoplasm and vacuoles to total Cu contents in the leaf and root cells decreased. The Cu-EDDS complex content in the leaf cell walls and cytoplasm accounted for 90.33% -89.95% and 9.67%-10.05% of the total complex contents in the leaf cells. Under the lower EDDS complex concentration conditions, all of the Cu-EDDS complex in root cells existed in the cell walls, and under the higher concentration of EDDS conditions, the Cu-EDDS complex was not only existed in the cell walls, but also in the cytoplasm and vacuoles. The results showed that the plasma membrane, under physiological conditions, could prevent Cu-EDDS complex from entering the cells.Cu accumulation mechanism induced by EDDS included several subsequent steps:(1) Combining EDDS with the Cu in soil forms Cu-EDDS complex and increases Cu bioavailability. (2) Higher concentration EDDS induces the membrane permeability of the root cells to increase. (3)Binding a small amount of Cu2+ions with anionic sites on the root cell walls leads Cu2+ions to remain in the roots. (4) A large number of Cu2+ ions are passively absorbed by roots from the "breaks" in the endodermis and the Casparian strips and "physiological barriers" destroyed by the toxic effects of EDDS into the xylem and transported to the above-ground parts as Cu-EDDS complex form. (5) A large amount of Cu in the shoots exists in cell walls as Cu-EDDS complex form, a small amount of Cu exists in cell walls or cytoplasm as other Cu compound form. Among these steps, the increase of soil soluble Cu contents and the membrane permeability of the root cells may play a critical role in shoot Cu accumulation process induced by EDDS; Cu-EDDS complex could not enter the cell membrane under physiological conditions. |
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