Complexation and Toxicity of Copper in Higher Plants. II. Different Mechanisms for Copper versus Cadmium Detoxification in the Copper-Sensitive Cadmium/Zinc Hyperaccumulator Thlaspi caerulescens (Ganges Ecotype)

The cadmium/zinc hyperaccumulator Thlaspi caerulescens is sensitive toward copper (Cu) toxicity, which is a problem for phytoremediation of soils with mixed contamination. Cu levels in T. caerulescens grown with 10 microm Cu(2+) remained in the nonaccumulator range (<50 ppm), and most individuals were as sensitive toward Cu as the related nonaccumulator Thlaspi fendleri. Obviously, hyperaccumulation and metal resistance are highly metal specific. Cu-induced inhibition of photosynthesis followed the "sun reaction" type of damage, with inhibition of the photosystem II reaction center charge separation and the water-splitting complex. A few individuals of T. caerulescens were more Cu resistant. Compared with Cu-sensitive individuals, they recovered faster from inhibition, at least partially by enhanced repair of chlorophyll-protein complexes but not by exclusion, since the content of Cu in their shoots was increased by about 25%. Extended x-ray absorption fine structure (EXAFS) measurements on frozen-hydrated leaf samples revealed that a large proportion of Cu in T. caerulescens is bound by sulfur ligands. This is in contrast to the known binding environment of cadmium and zinc in the same species, which is dominated by oxygen ligands. Clearly, hyperaccumulators detoxify hyperaccumulated metals differently compared with nonaccumulated metals. Furthermore, strong features in the Cu-EXAFS spectra ascribed to metal-metal contributions were found, in particular in the Cu-resistant specimens. Some of these features may be due to Cu binding to metallothioneins, but a larger proportion seems to result from biomineralization, most likely Cu(II) oxalate and Cu(II) oxides. Additional contributions in the EXAFS spectra indicate complexation of Cu(II) by the nonproteogenic amino acid nicotianamine, which has a very high affinity for Cu(II) as further characterized here

Biochemical and biophysical characterisation yields insights into the mechanism of TcHMA4, a Cd/Zn transporting ATPase purified from the hyperaccumulator plant Thlaspi caerulescens

AbstractTcHMA4 (GenBank no. AJ567384), a Cd/Zn transporting ATPase of the P1B-type (=CPx-type) was isolated and purified from roots of the Cd/Zn hyperaccumulator Thlaspi caerulescens. Optimisation of the purification protocol, based on binding of the natural C-terminal His-tag of the protein to a Ni-IDA metal affinity column, yielded pure, active TcHMA4 in quantities sufficient for its biochemical and biophysical characterisation with various techniques. TcHMA4 showed activity with Cu(2+), Zn(2+) and Cd(2+) under various concentrations (tested from 30nM to 10μM), and all three metal ions activated the ATPase at a concentration of 0.3μM. Notably, the enzyme worked best at rather high temperatures, with an activity optimum at 42°C. Arrhenius plots yielded interesting differences in activation energy. In the presence of zinc it remained constant (EA=38kJ⋅mol−1) over the whole concentration range while it increased from 17 to 42kJ⋅mol−1 with rising copper concentration and decreased from 39 to 23kJ⋅mol−1 with rising cadmium concentration. According to EXAFS the TcHMA4 appeared to bind Cd(2+) mainly by thiolate sulphur from cysteine, and not by imidazole nitrogen from histidine