Although copper (Cu) is required for plant growth and development, excessive levels of this element can be toxic to plants. Soil pollution and degradation take place when Cu is released into the environment through activities such as mining, discharge of "three wastes" (i.e., waste water, waste gas, and waste residue), and application of cupric fungicides. Medicago sativa, a perennial flowering plant native to Iran, has been cultivated as an important forage crop in many countries around the world. This species is reported to be a copper-hyperaccumulator, and studies suggest that it may be useful for phytoremediation.
To elucidate the mechanism of copper tolerance in M. sativa, changes in levels of chemical constituents of M. sativa treated with different concentrations of Cu solution were investigated using Fourier transform infrared spectroscopy (FTIR), an accurate, simple, efficient technique with high resolution.
After germination in a seed tray, M. sativa seedlings of uniform size and appearance were selected and transplanted into a hydroponics system. After pre-cultivation with one-quarter strength Hoagland's solution for one week, the seedlings were treated with different concentrations of Cu²⁺, i.e., 0, 1, 5, 20, and 100 mol/L. After 45 days of treatment with Cu²⁺ solution, roots, stems, and leaves were separated and dried to measure biomass and to detect changes in chemical constituent levels.
Although root, stem, and leaf biomass of M. sativa decreased slightly as concentrations of Cu²⁺ were increased, these changes were not found to be significant. After an initial decline, absorbance in roots of the dominant infrared band near 2924 cm^-1 exhibited an increasing trend. This result indicates that at low Cu²⁺ concentrations (<5 mol/L), organic acids secreted by M. sativa were able to chelate Cu²⁺, leading to a decrease in carboxylic acid O-H; at high Cu²⁺ concentrations (>5 mol/L), chelating activity decreased, which was followed by an increase in organic acids. Absorbance changes at 1381 cm^-1 mirrored those at 2924 cm^-1 as Cu²⁺ concentration was increased, indicating that fat levels inc reased after an initial decrease. The observed pattern of changes in fat concentration may have been due to increases in cell wall cation-exchange capacity that enhanced Cu resistance. In stems, absorbances of the dominant infrared bands near 2924, 1643, 1381, and 1064 cm^-1, which corresponded to organic acids, proteins and amino acids, fats, and carbohydrates, respectively, did not exhibit any obvious changes. In leaves, there were no obvious changes in absorbances of any dominant bands at low Cu²⁺ concentrations; at high Cu²⁺ concentrations, however, these absorbances fell after an initial rise. The observed trend may have been due to changes in levels of soluble sugar and soluble protein.
To summarize, roots of M. sativa are able to adjust organic acid content and to enhance cell wall cation-exchange capacity by accumulating Cu²⁺ and impeding transportation of Cu²⁺ from roots to shoots. As a result, shoots were effectively protected from Cu damage.