• heavy metals
• molecular biology
• phytoremediation
• tolerance
• translocation
• uptake

B. carinata cv. 079444 was selected, among nine different crop species, to remediate a multiple-contaminated area (As, Cd, Cu, Pb and Zn).
Since metals were poorly available, [S,S]-EDDS and NTA amendments were used to enhance B. carinata phytoextraction capacity.
With respect to NTA, EDDS improved both mobilisation from soil and accumulation in B. carinata shoots of Cu, Pb and Zn, whereas As and Cd were poorly extracted and accumulated.
One week after chelant addition, the DTPA-extractable metal concentrations in the polluted soil were lower in the EDDS treatment in comparison with the NTA one because of a faster biodegradation of metal-EDDS complexes.
In order to evaluate the possibility of a wild species-assisted phytoextraction, B. carinata plants were grown in succession to three wild species (Pinus pinaster, Plantago lanceolata and Silene paradoxa). The growth of the wild species in the multiple-polluted soil caused an enhancement of the DTPA-exchangeable metals, the pattern of metal mobilisation being species-specific. A 2-unit decrease of the soil pH following the cultivation of the three wild species was detected, and the analysis of organic compounds, in terms of flavonoids, organic and phenolic acids, exuded by Pinus pinaster, Plantago lanceolata and Silene paradoxa was performed in order to explain the species-specific patterns of metals mobilisation. In agreement with the available metal amounts in soil, an enhancement of B. carinata capacity to accumulate and translocate Cd, Cu, Pb and Zn was detected with the values of metals concentrations in shoots approaching or exceeding (as in the case of Cd) those detected following chelators (NTA or EDDS) amendments.
The uptake mechanisms of different arsenic and copper species were studied.
Arsenite showed linear influxes in time- and concentration-dependent uptake kinetics. For arsenate and copper, uptake kinetics showed saturation in time-dependent plot; biphasic patterns were observed as function of their external concentration. The linear uptake mechanisms, when observed, were likely due to As(III)-, As(V)- or Cu(II)-induced damages at the plasma membrane level. Competitive inhibition of uptake with phosphate showed that arsenite and arsenate were taken up by different uptake systems.
The effects of Cu-NTA- and Cu-EDDS2- complexes on the copper uptake by excised B. carinata roots were investigated. Typical ¡§enzyme saturation¡¨ curves were found with the anionic complexes accumulated at a slower rates than the positive charged hexaquocopper (II) cation, [Cu(H2O)6]2+.
To test the effect of both chelators in the uptake and translocation of copper during time, long-term experiments of accumulation of Cu, NTA and EDDS were performed as well. Results confirmed, doubtlessly, the involvement of the two chelators in the enhanced Cu translocation.
The effect of high (2.5 and 5 ƒÝM) and low (0 ƒÝM) external copper concentrations on amino acids amounts and copper speciation in the xylem sap of B. carinata plants was studied. When plants were exposed to high Cu concentrations in the nutrient solution, histidine (His), threonine (Thr), glutamine (Gln), proline (Pro), methionine (Met) and glycine (Gly) showed the greatest increases. In copper starvation conditions, methionine (Met), nicotianamine (NA), glutamine (Gln) and threonine (Thr) concentrations increased. His and NA were found to be the most important Cu xylematic transporters in copper excess and deficiency conditions, respectively.
Finally, to enhance the efficiency of metal accumulation by plants, the genetic engineering approach was evaluated. The model plant Nicotiana tabacum SR1 was transformed with the MT2b gene from A. thaliana in order to improve its As(III) tolerance, sequestration and accumulation.
MT2b-transformed tobacco plants, if compared to wild type, showed no significant differences in As(III) tolerance to mildly toxic levels of As(III) exposure (3 and 6 ƒÝM), but they showed to be more sensitive to more toxic As(III) levels (12 ƒÝM). The insertion of AtMT2b gene in tobacco plants did neither increase As(III) accumulation both on a dry biomass and on a plant basis. The higher resistance of the wild type to As(III) is therefore not due to a reduction of the net As(III) influx.