Excess copper predisposes photosystem II to photoinhibition in vivo by outcompeting iron and causing decrease in leaf chlorophyll

Abstract

Photoinhibition of photosystem II was studied in vivo with bean (Phaseolus vulgaris) plants grown in the presence of 0.3 (control), 4, or 15 microM Cu(2+). Although photoinhibition, measured in the presence of lincomycin to block concurrent recovery, is faster in leaves of Cu(2+)-treated plants than in control leaves, thylakoids isolated from Cu-treated plants did not show high sensitivity to photoinhibition. Direct effects of excess Cu(2+) on chloroplast metabolism are actually unlikely, because the Cu concentration of chloroplasts of Cu-treated plants was lower than that of their leaves. Excess Cu in the growth medium did not cause severe oxidative stress, collapse of antioxidative defenses, or loss of photoprotection. Thus, these hypothetical effects can be eliminated as causes for Cu-enhanced photoinhibition in intact leaves. However, Cu treatment lowered the leaf chlorophyll (Chl) concentration and reduced the thylakoid membrane network. The loss of Chl and sensitivity to photoinhibition could be overcome by adding excess Fe together with excess Cu to the growth medium. The addition of Fe lowered the Cu(2+) concentration of the leaves, suggesting that Cu outcompetes Fe in Fe uptake. We suggest that the reduction of leaf Chl concentration, caused by the Cu-induced iron deficiency, causes the high photosensitivity of photosystem II in Cu(2+)-treated plants. A causal relationship between the susceptibility to photoinhibition and the leaf optical density was established in several plant species. Plant species adapted to high-light habitats apparently benefit from thick leaves because the rate of photoinhibition is directly proportional to light intensity, but photosynthesis becomes saturated by moderate light.

Figure 1

Photoinhibition of oxygen evolution in thylakoids isolated from control bean leaves (○) and from leaves of bean plants grown in the presence of 15 μm Cu²⁺ (▵). Isolated thylakoids were illuminated at 1,000 (white symbols, dashed line) or 2,000 μmol photons m⁻² s⁻¹ (black symbols, solid line).

Figure 2

A, Oxyblot visualizing carbonyl groups in thylakoid proteins. The thylakoids were isolated from control bean plants (0.3 μm Cu²⁺) and from bean plants grown at 4 and 15 μm Cu²⁺. Each sample contained 7.5 μg of soluble protein. The arrows mark the positions of M_r standards. B, The amount of malondealdehyde (MDA) of the thylakoids isolated from the bean leaves, measured with the thiobarbituric acid method. C, The amount of total glutathione determined from the bean leaves; D, the ratio of GSH to GSSG. In B through D, white, hatched, and black bars correspond to plants grown in the presence of 0.3 (control), 4, and 15 μm Cu²⁺ in growth medium, respectively. Each bar represents the mean of three independent experiments, and the error bars show se.

Figure 3

A, Activities of SOD, APX, and GR measured from the first trifoliate leaves of bean plants after 2 weeks of growth in the presence of 0.3 μm Cu²⁺ (control plants; white bars), 4 μm Cu²⁺ (hatched bars), or 15 μm Cu²⁺ (black bars). B, The coefficient of nonphotochemical (q_N, circles) and photochemical (q_Q, squares) quenching of Chl fluorescence in leaves of control beans (white symbols) and leaves of bean plants grown in the presence of 4 μm Cu²⁺ (black symbols). Fluorescence was measured with a PAM fluorometer after 5 min of illumination at each PPFD, and far-red illumination was used to measure F₀ ' after each white light illumination period. Each data point shows the mean of four independent experiments, and the error bars show se.

Figure 4

Electron micrographs of bean chloroplasts. A, Chloroplasts of a control plant. B, Chloroplasts of a plant grown in the presence of 4 μm Cu²⁺. C, Chloroplasts of a plant grown in the presence of 15 μm Cu²⁺. Black bar = 2 μm. The upper left corner of each image shows a magnification of a grana stack; white bar = 0.2 μm.

Figure 5

Dependence of photoinhibition on Chl concentration. Lincomycin-treated leaves of Sinapis alba (⋆), Alliaria petiolata (□), Plantago major (▵), Tilia platyphyllos (⋄), Alchemilla vulgaris (▿), and Aesculus hippocastanum (○) were illuminated at the PPFD of 1,500 μmol m⁻² s⁻¹. The k_PI values are based on measurements of oxygen evolution. The k_PI values of control bean leaves (●) and from leaves of beans grown in the presence of 4 μm Cu²⁺ (▪) or 15 μm Cu²⁺ (▴) were multiplied by 1.5 to compensate for the lower PPFD (1,000 μmol m⁻² s⁻¹) used to obtain these values. The bean data is from Pätsikkä et al. (1998). The line is the best fit to Equation 1, each data point corresponds to an independent experiment, and the error bars, drawn if larger than the symbol, indicate se of the curve fit.

Figure 6

Model of the in vivo mechanism by which excess Cu makes PSII more susceptible to photoinhibition. The main primary effect of excess Cu is Fe deficiency, which causes the metabolic disturbances leading to reduction of the Chl concentration in leaves. Leaves with low Chl concentration are sensitive to photoinhibition. Cu²⁺-Induced oxidative stress may enhance the symptoms of Fe deficiency.