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CONCLUSIONS. DD
DDIFFERENT IFFERENT IFFERENT IFFERENT PPPPHYTOEXTRACTION HYTOEXTRACTION HYTOEXTRACTION HYTOEXTRACTION TTTTECNIQUES OF ECNIQUES OF ECNIQUES OF ECNIQUES OF HHHHEAVY EAVY EAVY EAVY MMMMETALSETALSETALSETALS
The present study showed that, in comparison to eight other crop plant species, B.
carinata was the most efficient in accumulating metals in shoots. EDDS proved to be more effective than NTA in the phytoextraction of a soil contaminated by As, Cd, Cu, Pb and Zn. The treatment with 5 mmol EDDS kg-1 soil resulted in accumulation of 157, 122 and 129 mg kg-1 dry weight of Cu, Pb and Zn, respectively, which was about 2-fold that of the corresponding NTA treatments. The increased translocation of metals following chelators addition is likely due to the damages that these synthetic chelators cause on the physiological barriers of roots. This allows the influx of the negatively charged complexes metal-chelator through via non-selective apoplastic pathways.
Long-term experiments of accumulation of Cu, NTA and EDDS in roots and shoots of B. carinata confirmed the chelator-induced plasma membrane destabilisation. Since this destabilisation arose after about 12 h of incubation, the principal factor for inducing membrane damages seems to be the time of contact between the chelators and the roots.
In fact, in the uptake kinetic studies (incubation time 30 min) of Cu-NTA- and Cu- EDDS2- complexes no plasma membrane damages were detected. However, the presence of chelators (NTA or EDDS) changed the pattern of copper uptake. When chelators were added to the treatment solutions, the biphasic uptake mechanism detected for free Cu2+ was supplanted by simple Michaelis-Menten uptakes. These differences among the kinetic patterns of free Cu2+, Cu-NTA- and Cu-EDDS2- can be better explained in terms of an inhibition of the uptake, due to the presence in treatment solution of the two chelators, instead of in terms of chelators-induced membrane damages.
The selection of a contact period longer than 12 h in the phytoextraction experiments in soil have likely allowed the chelators to cause damages at the root level and, therefore, to be easily transported as metal-complexes to the shoots. The high biodegradability of both chelators, EDDS in particular (half life time 7 days), reduced the risks associated
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with the leaching of metals to the groundwater during this relatively long period of amendment.
Since the biodegradation of chelators is always related to the experimental conditions, an accurate and exhaustive risk assessment study should be performed in each case. For this reason, the long-term goal of phytoremediation is doubtlessly to avoid the addition of synthetic substances to the soil. In order to reach this purpose, two totally safe approaches for phytoremediation were studied.
Firstly, different plant species were combined in succession, growing in the multiple- contaminated soil three non-accumulator species (Pinus pinaster, Plantago lanceolata and Silene paradoxa) before B. carinata. This combination gave good results with the metal mobilisation in soil and metal accumulation in the harvestable part of B. carinata approaching or exceeding the values obtained using the well-known chelator-assisted phytoextraction technique. The study of the organic substances exuded by the three non- accumulator species confirmed the role of this compounds in the improvement of the phytoextraction capacity of B. carinata.
Second, the insertion of the MT2b gene from Arabidopsis thaliana into the genome of the model plant Nicotiana tabacum was performed in order to obtain a plant with enhanced metal phytoextraction capacity.
Unfortunately, the insertion of the AtMT2b gene didn’t show any promising results in terms of tolerance and accumulation of As(III). A possible depletion in plant glutathione and phytochelatins content leading to a reduced capacity of those plants to bind As(III) and sequester it in the vacuolar cellular compartment as As(III)-GS3 or As(III)-PCs could come into play. Investigations about the glutathione and phytochelatins content of Nicotiana tabacum wild type and AtMT2b-transformed plants might confirm this hypothesis. Investigations about the role of the AtMT2b gene in the tolerance of the other metals present in the multiple-polluted soil (Cd, Cu, Pb and Zn) are also being performed. Depending on the results, the development of a transformation protocol for B. carinata will be taken into consideration.
Arsenic and copper were chosen, among the five metals present in the multiple-polluted site, to study their uptake mechanisms by B. carinata roots. As arsenic was present in
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the site in its inorganic chemical forms (arsenate and arsenite), the uptake of both species was investigated.
The short-term concentration-dependent uptake kinetics obtained showed that the uptakes of arsenate and copper (as hexaquocopper(II) cation) were both active processes until those elements didn’t cause significant damages to the plasma membranes of B.
carinata roots. While it is reasonable to suppose that copper is transported across the plasmalemma with an active process, being an essential element for plants, the interpretation of the results obtained for arsenate requires caution. Competitive inhibition of uptake with phosphate justified the active uptake of arsenate into the plant root cells being this toxic metalloid taken up through the phosphate channels. Arsenite was taken up by B. carinata roots following an osmoregulated pathway likely due to the damages that this trivalent metalloid caused on roots plasma membranes also at very low concentrations.
Increases in concentrations of Cu in nutrient solutions (2.5 and 5 µM CuSO4) induced the synthesis of certain amino acids which include histidine, proline, glycine and threonine which have high association constants with Cu. Surprisingly, nicotianamine, that have the highest association constant for Cu, didn’t show any significant difference if compared to the control (0.12 µM CuSO4). However, as far as the stability constants are concerned, histidine could compete with nicotianamine as a ligand for Cu. Copper complexation studies indicated that about 63% of all copper transported in xylem sap of B. carinata was present as negatively charged complexes involving amino acids. The concentration of histidine in B. carinata xylem sap can account for all the bound Cu carried in the xylem. Confirmation of the importance of this Cu ligand came from comparative studies between B. carinata xylem sap and simulated saps containing single amino acids or combinations of them.
In copper starvation conditions, the synthesis of methionine, nicotianamine, glutamine and threonine was induced indicating that the B. carinata response in terms of amino acids production is stress-specific. The pH sensitive Cu-binding signature curves of Brassica carinata xylem sap and simulated saps containing single amino acids showed that nicotianamine is likely the most important copper ligand in xylem exudates of B.
carinata in condition of copper starvation. Since nicotianamine is an intermediate in
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mucigenic-acids (MAs) biosynthesis, it is not unreasonable to think that it is involved in internal copper transport when the plant is in metal deficiency rather than in metal excess.
