**4. Biochemical methods**

The biochemical parameters, stress markers such as phenols, proline, malondialdehyde (MDA), thiol groups, hydrogen peroxide (H2O2), glutathione, and leaf pigments (chlorophyll a and b) were determined to analyze the physiological stage of the plants and to perform a comparative analysis with the results from the spectral remote sensing methods. All stress markers were measured using a spectrophotometer Multiskan Spectrum (Thermo Electron Corporation). Fresh leaf materials (0.2 g) of soybean plants were homogenized at 4 ºC in 0.1% cold trichloroacetic acid. The homogenates were centrifuged at 15000 g for 30 min. Then the supernatants obtained were used for determination of the stress markers after applying different methods.

Phenols and proline are important protective components of the plant cells and they accumulate when cells are in stress conditions. The antioxidant properties of phenols are mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donators and singlet oxygen quenchers (Rice-Evans et al., 1997; Khan et al., 2002). Plants generally accumulate some of osmolytes such as proline under salt and drought stress (Delauney & Verma, 1993), which protect the proteins against denaturation and also act as osmotic balancing agents (Sivakumar et al., 2000). Content of total free phenols was measured according to the method developed by Swain and Goldstein (1959) using Folin - Ciocalteau reagent. Caffeine acid was used as a standard. The absorbance was registered at 725 nm. Plants generally accumulate some of osmolytes such as proline under salt and drought stress, which protect the proteins against denaturation and also act as osmotic balancing agents (Chadalavada et al., 1994; Kavi Kishor et al., 2005). Proline content was determined by measuring the absorbance at 520 nm after reaction with a mixture glacial acetic acid and ninhydrin according the method of Bates et al., (1973). Proline concentration in the samples was determined from the standard curve calibrated with different concentrations of the standard proline.

Hydrogen peroxide is known to damage various cellular components and evoke structural modifications of proteins, lipids and DNA under stress (Halliwell & Gutteridge, 2002). Hydrogen peroxide and malondialdehyde contents are routinely estimated together with electrolyte leakage measurements to assess the extent of oxidative stress. The injurious impact of reactive oxygen species on the membranes of cells is realized by lipid peroxidation. The basic damage products by this process are aldehydes and mainly MDA. The accumulation of MDA is an especially sensitive marker of stress. For determination of the amount of MDA as a final product of lipid peroxidation, the method of Kramer et al. (1991) was used. The absorbance was measured at 532 nm and 600 nm. The content of endogenous free thiol groups was determined using the Elman's reactive according to the method of Edreva & Hadjiska, (1984). Hydrogen peroxide absorbance was measured at 390 nm after reaction with KJ according to Alexieva et al., (2001). Content of Н2О2 was calculated using the standard curve with known amount of KJ. Glutathione content was measured according to Gronwald et al., (1998).

Compounds bearing a free thiol groups (-SH), such as low molecular cell metabolites like glutathione, as well as a number of enzymes, which are active only in a reduced state, play a key role in important cellular functions, and the massive oxidation of -SH groups could be regarded as an aspect of oxidative toxicity (Haugard, 2000). The relationship between the SH-state in plants and their resistance to various stress factors is well known. Glutathione is major fraction of the SH pool in the cells (Haugard, 2000; Noctor et al., 2002).

Pigment contents (chlorophyll a and b) were calculated after the extraction of leaf material with 80% acetone according to the method of Arnon (1949). The extinction of chlorophyll a and chlorophyll b was determined at 663 nm and 845 nm using a spectrometer Specol 11.
