Complexation and toxicity of copper in higher plants. I. Characterization of copper accumulation, speciation, and toxicity in Crassula helmsii as a new copper accumulator

Abstract

The amphibious water plant Crassula helmsii is an invasive copper (Cu)-tolerant neophyte in Europe. It now turned out to accumulate Cu up to more than 9,000 ppm in its shoots at 10 microm (=0.6 ppm) Cu(2+) in the nutrient solution, indicating that it is a Cu hyperaccumulator. We investigated uptake, binding environment, and toxicity of Cu in this plant under emerged and submerged conditions. Extended x-ray absorption fine structure measurements on frozen-hydrated samples revealed that Cu was bound almost exclusively by oxygen ligands, likely organic acids, and not any sulfur ligands. Despite significant differences in photosynthesis biochemistry and biophysics between emerged and submerged plants, no differences in Cu ligands were found. While measurements of tissue pH confirmed the diurnal acid cycle typical for Crassulacean acid metabolism, Delta(13)C measurements showed values typical for regular C3 photosynthesis. Cu-induced inhibition of photosynthesis mainly affected the photosystem II (PSII) reaction center, but with some unusual features. Most obviously, the degree of light saturation of electron transport increased during Cu stress, while maximal dark-adapted PSII quantum yield did not change and light-adapted quantum yield of PSII photochemistry decreased particularly in the first 50 s after onset of actinic irradiance. This combination of changes, which were strongest in submerged cultures, shows a decreasing number of functional reaction centers relative to the antenna in a system with high antenna connectivity. Nonphotochemical quenching, in contrast, was modified by Cu mainly in emerged cultures. Pigment concentrations in stressed plants strongly decreased, but no changes in their ratios occurred, indicating that cells either survived intact or died and bleached quickly.

Figure 1.

Photographs of plants (emerged stock culture) after 1 week of treatment. Top panels, Emerged treatments; bottom panels, submerged treatments; left panels, control (0.1 μm Cu²⁺); right panels, stressed with 10 μm Cu²⁺.

Figure 2.

Changes in pigment contents in C. helmsii leaves as a result of emerged versus submerged growth and Cu stress. The data are averages and se of three independent experiments. Treatment labels are as follows: E, experiment (treatment) in emerged state; S, experiment in submerged state; Cu, treated with 10 μm Cu²⁺; Ct, control (0.1 μm Cu²⁺).

Figure 3.

Results of pH measurements of C. helmsii leaves for detecting circadian changes of acid content indicative of CAM. The lines between the morning and evening measuring points are only a visual aid for making it easier to see which pairs of points belong together; they do not imply that there would be a linear decrease of pH over the day. The data are averages of two independent experiments. Treatment labels are as follows: e, emerged stock culture; s, submerged stock culture; E, experiment (treatment) in emerged state; S, experiment in submerged state; Cu, treated with 10 μm Cu²⁺; Ct, control (0.1 μm Cu²⁺).

Figure 4.

Changes in the biophysics of photosynthesis as a result of emerged versus submerged growth and Cu stress, revealed by Chl fluorescence parameters. All samples were measured in the FKM at an actinic irradiance of 120 μmol m⁻² s⁻¹. See “Materials and Methods” for details of the measuring protocol, including timing of events. The data are averages of two independent experiments. Treatment labels are as follows: E, experiment (treatment) in emerged state; S, experiment in submerged state; Cu, treated with 10 μm Cu²⁺; Ct, control (0.1 μm Cu²⁺). A, Selected parameters. Indices of sections of the measuring protocol are as follows: “_d” stands for “dark adapted,” “_i” stands for “irradiated with actinic light,” and “_r” stands for “relaxation period after actinic irradiance.” The numbers in the index sequentially number the SIPs in the respective protocol section. A short explanation of individual fluorescence kinetic parameters is provided in Table I; for details, see reviews on Chl fluorescence kinetics or Küpper et al. (2007a). Top row, Basic parameters of fluorescence yield in nonactinic (F₀) and supersaturating (F_m) light; second row, parameters measuring photochemical activity (F_v/F_m and Φ_PSII); third row, parameters measuring NPQ; bottom row, light saturation as measured by F_p in relation to F_m with correction of F₀ effects. B, Typical examples of complete fluorescence kinetic measurements. See “Materials and Methods” for details of the measuring protocol, including timing of events. The black bars at the bottom of the panels indicate periods of the measurement when actinic light is switched off, and the white bars symbolize the time period of actinic irradiance. The arrows indicate the positions of the SIPs.

Figure 5.

Spectra of in situ x-ray absorbance measurements (Cu-K edge) of frozen-hydrated C. helmsii tissues. Treatment labels are as follows: Ef, Fermi energy (Rehr and Albers, 1990); E, experiment (treatment) in emerged state; S, experiment in submerged state; Cu, treated with 10 μm Cu²⁺; Ct, control (0.1 μm Cu²⁺). Top, Normalized EXAFS and fit with theoretical model. Bottom, Fourier transform of the EXAFS and fits with theoretical model (done on normalized EXAFS).