Copper Toxicity in Plants

Prior to the identification of copper as a micronutrient, it was regarded as a plant poison (7). Therefore, no discussion of copper toxicity can rightfully begin without mention of its use as a fungicide. In 1882, botanist Pierre-Marie-Alexis Millardet developed a copper-based formulation that saved the disease-ravaged French wine industry (68). Millardet’s observation of the prophylactic effects against downy mildew of grapes by a copper sulfate–lime mixture led to the discovery and development of Bordeaux mixture [CuSO₄.5H₂O⁺Ca(OH)₂]. Incidentally, this copper sulfate–lime mixture had been sprinkled on grapevines along the roadways for decades to prevent the stealing of grapes. The observation that Bordeaux sprays sometimes had stimulating effects on vigor and yield led to the experimentation that eventually proved the essentiality of copper as a plant micronutrient (7). It is likely that copper fungicides corrected many copper deficiencies before copper was identified as a required element (69).

The currently accepted theory behind the mode of action of copper as a fungicide is its nonspecific denaturation of sulfhydryl groups of proteins (70). The copper ion is toxic to all plant cells and must be used in discrete doses or relatively insoluble forms to prevent tissue damage (70). There are a multitude of copper-based fungicides and pesticides available to agricultural producers. Overuse or extended use of these fungicides in orchards and vineyards has produced localized soils with excessive copper levels (71).

The two general symptoms of copper toxicity are stunted root growth and leaf chlorosis. For ryegrass (Lolium perenne L.) seedlings in solution culture, the order of metal toxicity affecting root growth was Cu⁺Ni + Mn + Pb + Cd + Zn + Al + Hg + Cr + Fe (72). This order is supported by earlier experiments with Triticum spp., white mustard (Sinapis alba L.), bent grass (Agrostis spp. L.), and corn (72). Stunted roots are characterized by poor development, reduced branching, thickening, and unusual dark coloration (7,14,72,73). Small roots and apices of large roots of spinach turned black in response to 160 �M Cu in nutrient solution culture (73). Root growth was decreased progressively in corn when plants were exposed to 10^-5, 10^-4, 10^-3 M Cu²⁺ in solution culture (14). However, due to the complexity of cell elongation in roots and influences of hormones, cell wall biosynthesis, and cell turgor, few research studies have defined the effect of copper on root growth (74).

Copper-induced chlorosis, oftentimes resembling iron deficiency, reportedly occurs due to Cu⁺ and Cu²⁺ ion blockage of photosynthetic electron transport (75). Chlorophyll content of spinach leaves was decreased by 45% by treatment of 160 �M Cu in solution culture over control treatment (73). Increasing Cu²⁺ exposure to cucumber cotyledon and leaf tissue extracts decreased the amount of UV-light absorbing compounds (76). Chlorosis of bean (Phaseolus vulgaris L.) and barley was observed with copper toxicity (77,78). Energy capture efficiency and antenna size were decreased in spinach leaves exposed to toxic levels of copper (73). Copper toxicity symptoms of oregano (Origanum vulgare L.) leaves included thickening of the lamina and increases in number of stomata, glandular, and nonglandular hairs, as well as decreases in chloroplast number and disappearance of starch grains in chloroplasts of mesophyll cells (79). Copper ions also may be responsible for accelerating lipid peroxidation in chloroplast membranes (75).

In the photosynthetic apparatus, the donor and acceptor sites of Photosystem II (PSII) are sensitive to excess Cu²⁺ ions (80). The suggested sites of Cu²⁺ inhibition on the acceptor side of PSII are the primary quinone acceptor QA (81,82), the pheophytin-QA-Fe region (83), the non-heme Fe (82,84), and the secondary quinone acceptor QB (85). On the donor side of PSII, a reversible inhibition of oxidation of TyrZ (oxidation-reduction active tyrosine residue in a protein component of PSII) has been observed by Schr�der et al. (86) and Jegersch�ld et al. (81). However, Cu²⁺ ions in equal molar concentration to the number of PSII reaction centers stimulated oxygen evolution nearly twofold, suggesting that Cu²⁺ may be a required component of PSII (80). Substitution for magnesium in the chlorophyll heme by copper has been observed in brown and green alga under high or low irradiance during incubation at 10 to 30 �M CuSO₄ (67). High Cu²⁺ tissue concentrations inhibited oxygen evolution and quenched variable fluorescence (87). Brown and Rattigan (88) reported rapid and complete oxygen production in an aquatic macrophyte (Elodea canadensis Michx.) in response to copper toxicity. In fact, E. canadensis has been suggested to be a good biomonitor of copper levels in aquatic systems (89).

Excess heavy metals often alter membrane permeability by causing leakage of K⁺ and other ions. Solution culture experiments noted that 0.15 �M CuCl₂ decreased hydrolytic activity of H⁺-ATPase in vivo in cucumber roots, but stimulated H⁺ transport in corn roots (90). During these experiments, Cu²⁺ also inhibited in vitro H⁺ transport through the plasmalemma in cucumber roots but stimulated transport in corn roots (90). Copper toxicity also can produce oxidative stress in plants. Increased accumulation of the polyamine, putrescine, was detected in mung bean (Phaseolus aureus Roxb.) after copper was increased in solution culture (91). Fifteen-day-old wheat (Triticum durum Desf. cv. Cresco) roots exhibited a decrease in NADPH concentrations from 108 to 1.8 nmol g^-1, a 23% increase in glutathione reductase activity,and a 43-fold increase in ascorbate over control plants in response to 150 �M Cu in solution culture after a 168-h exposure (94).

In soil, copper toxicity was observed with upland rice (Oryza sativa L.) at an application of 51 mg Cu kg^-1 to the soil, common bean at 37 mg kg^-1, corn at 48 mg kg^-1, soybean at 15 mg kg^-1, and wheat (Triticum aestivum L.) at 51 mg kg^-1 (93). An adequate copper application rate was 3 mg kg^-1 for upland rice, 2 mg kg^-1 for common bean, 3 mg kg^-1 for corn, and 12 mg kg^-1 for wheat. In this study, an adequate soil test for copper was 2 mg kg^-1 for upland rice, 1.5 mg kg^-1for common bean, 3 mg kg^-1 for corn, 1 mg kg^-1 for soybean, and 10 mg kg^-1 for wheat, when Mehlich-1 extracting solution was used. The toxic level for the same extractor was 48 mg kg^-1 for upland rice, 35 mg kg^-1 for common bean, 45 mg kg^-1 for corn, 10 mg kg^-1 for soybean, and 52 mg kg^-1 for wheat. Copper (Cu²⁺) significantly inhibited growth of radish seedlings at 1 �M in solution culture (94). Addition of supplemental iron to nutrient solution culture lessened the effects of artificially induced copper toxicity in spinach (73). At 10 �M, Cu in the nutrient solution decreased epicotyl elongation and fresh weight of mung bean, but increasing the calcium concentration in the solution to 5 �M improved growth (91). Wheat net root elongation, in relation to the original length, was only 13% in solution culture in response to 1.75 �M Cu²⁺ as Cu(NO₃)₂, but additions of 240 �M malate with the Cu(NO₃)₂ increased root elongation to 27%; addition of 240 �M malonate increased root to 67%, and 240 �M citrate increased growth to 91%, indicating the potential of these organic ligands to complex Cu²⁺ and to lessen its toxicity (95).