Use of micro-PIXE to determine spatial distributions of copper in Brassica carinata plants exposed to CuSO4 or CuEDDS.

A better understanding of the mechanisms that govern copper (Cu) uptake, distribution and tolerance in Brassica carinata plants in the presence of chelators is needed before significant progress in chelate-assisted Cu phytoextraction can be made. The aims of this study were therefore to characterise (S,S)-N,N'-ethylenediamine disuccinic acid (EDDS)-assisted Cu uptake, and to compare the spatial distribution patterns of Cu in the roots and leaves of B. carinata plants. The plants were treated with 30 µM or 150 µM CuSO(4) or CuEDDS in hydroponic solution. Quantitative Cu distribution maps and concentration profiles across root and leaf cross-sections of the desorbed plants were obtained by micro-proton induced X-ray emission. In roots, the 30 µM treatments with both CuSO(4) and CuEDDS resulted in higher Cu concentrations in epidermal/cortical regions. At 150 µM CuSO(4), Cu was mainly accumulated in root vascular bundles, whereas with 150 µM CuEDDS, Cu was detected in endodermis and the adjacent inner cortical cell layer. Under all treatments, except with a H(+)-ATP-ase inhibitor, the Cu in leaves was localised mainly in vascular tissues. The incubation of plants with 150 µM CuEDDS enhanced metal translocation to shoots, in comparison to the corresponding CuSO(4) treatment. Inhibition of H(+)-ATPase activity resulted in reduced Cu accumulation in 30 µM CuEDDS-treated roots and 150 µM CuEDDS-treated leaves, and induced changes in Cu distribution in the leaves. This indicates that active mechanisms are involved in retaining Cu in the leaf vascular tissues, which prevent its transport to photosynthetically active tissues. The physiological significance of EDDS-assisted Cu uptake is discussed.

The influence of EDDS on the metabolic and transcriptional responses induced by copper in hydroponically grown Brassica carinata seedlings.

To improve the knowledge about the use of plants for the removal of toxic metals from contaminated soils, metabolic and transcriptional responses of Brassica carinata to different forms of copper (Cu) were studied. Two-week-old hydroponically grown seedlings were exposed for 24 h to 30 µM CuSO4 or CuEDDS. CuSO4 appeared to be more toxic than CuEDDS as roots showed higher levels of thiobarbituric acid reactive substances (TBARS) and increased relative leakage ratios (RLR), although the superoxide dismutase (SOD, EC 1.15.1.1) activity increased following both exposures. In CuSO4-exposed seedlings the higher toxicity was underlined by increased transcription of lipoxygenases (EC 1.13.11.12) and NADPH oxidases (EC 1.6.99.6) and by the higher Cu accumulation in both tissues compared to CuEDDS exposure. The presence of EDDS increased Cu translocation, which resulted 5-times higher than when exposed to CuSO4. Decreases in catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) activities together with increases of reduced glutathione (GSH) and tocopherols and a reduction of lipoic acid (LA) were observed in roots of CuSO4-exposed seedlings. On the contrary, CuEDDS exposure induced a general increase in enzyme activities and decreases in ascorbate (AsA) and tocopherol levels. In the primary leaves, in both exposures Cu differently affected the oxidative stress responses indicating that the cellular redox balance was anyway maintained. EDDS plays a crucial role in B. carinata tolerance to oxidative stress induced by Cu and might be proposed to improve the efficiency of Cu phytoextraction.

Uptake and translocation of CuEDDS complexes by Brassica carinata.

The knowledge of the mechanisms that underlie metal complex uptake may lead to the development of new strategies for enhancing metal phytoextraction. As metals such as copper are actively taken up by roots, by inhibiting the proton driving force it is possible to obtain preliminary indications on the metal complex uptake mechanism. For this, Cu, EDDS, and Cu-EDDS uptake kinetics of Brassica carinata excised roots incubated in 30 and 150 microM solutions of either the metal, the chelant, and the complex were determined in the presence or not of the ATPase inhibitor vanadate. Following both Cu and CuEDDS treatments, metal uptake was negatively influenced by vanadate, whereas EDDS uptake did not, suggesting that Cu and the chelant did not enter the roots in their complexed form but by two different routes. The incubation in the same solutions of B. carinata intact plants showed that, differently from Cu, EDDS was largely translocated to shoots, but its low concentration resulted in a Cu to EDDS molar ratio ranging from 2 to 4 depending on metal complex concentration in the solution confirming that the uptake pathways of the two compounds were different.

Copper excess triggers phospholipase D activity in wheat roots.

Wheat seedlings (Triticum durum Desf.) were incubated in 100muM Cu(2+) for different periods of time ranging from 1min up to 16h. Following metal addition a rapid intake of copper ions into the roots was observed. Cu(2+) induced an accumulation of both phosphatidic acid and phosphatidylbutanol within 1min of incubation, the latter indicating a very rapid induction of phospholipase D (PLD) activity. The highest PLD stimulation was detected after 2h from copper addition and decreased almost to the initial value at increasing times. Cycloheximide treatment of roots lowered phosphatidylbutanol accumulation because of a reduced PLD activity. The expression profile of a T. durum putative PLD-encoding gene showed a peak after 1h of treatment as well, indicating that enhanced gene expression contributed to the increase in PLD activity. In the absence of copper ions, roots treated with the G protein activator mastoparan showed increases in phosphatidic acid and phosphatidylbutanol similar to those detected with the metal. PLD activity was also stimulated by cholera toxin. Two putatively G protein alpha subunit encoding sequences were isolated and no significant differences in transcription activity following Cu(2+) addition were observed. In copper-treated roots an early production of superoxide generated both by total and membrane-bound NADPH oxidase occurred. The G protein inhibitor suramin as well as the PLD antagonist 1-butanol abolished copper-induced superoxide production.