Phytotoxicity is a term used to describe the degree of toxic effect by a compound on the growth of plants. It is caused by a wide variety of compounds, including heavy metals, salinity, pesticides, phytotoxins or allelopathy. The sensitivity of plants to heavy metals depends on an interrelated network of physiological and molecular mechanisms such as uptake and accumulation of metals from cytoplasm to extra cellular exudates and cell wall constituents; efflux of heavy metals from cytoplasm to extra nuclear compartments including vacuoles; complexation of heavy metal ions inside the cell by various substances, for example, organic acids, phytochelatins, amino acids and metallothioneins; accumulation of osmolytes and induction of antioxidative enzymes; activation or modification of plant metabolism to allow adequate functioning of metabolic pathways and rapid repair of damaged cell structures [1]. Cadmium (Cd), being highly toxic metal pollutants of soils, which inhibits the root and shoot growth (yield), affects nutrient uptake. The application of sewage sludge, waste and cadmium containing fertilizers causes the increase of cadmium content in soils. As green gram is a stress sensitive legume, the present investigation was undertaken to analyze whether cadmium produces oxidative stress during early stages of seedling development.

Sample collection and preparation

The healthy seeds of Green gram (Vigna radiata linn) were collected from Tamil Nadu Agricultural University (TNAU), Coimbatore. The seeds (3 sets of 1 gram each) were soaked in 10, 50 μM solution of Cadmium chloride overnight. Then the seeds were sown in the earthern pots and allowed to grow naturally for a week. Every alternate day they were watered with distilled water in the green house. At the 7^th day seedlings were removed, used for the content of carbohydrate, protein, chlorophyll and superoxide dismutase, catalase and peroxidase.

Estimation of total carbohydrate: The total carbohydrate was estimated by anthrone method [2].

Reagents

1. 2.5N- HCl

2. Anthrone reagent

500 mg of the leaves was weighed and ground well with a pestle and mortar in 5-10 ml of 80% ethanol. The supernatant was collected and 0.5, 1 ml aliquots were taken for analysis. Standard solution was prepared by taking 0, 0.2, 0.4, 0.6, 0.8 and 1 ml of the working standard (glucose) as blank. The volume was made to 1 ml in all the tubes including the sample tubes by adding distilled water. Then 4 ml of anthrone reagent was added and was heated in a boiling water bath for 10 minutes and made to cool. Green to dark green colour was recorded at 630 nm. A standard graph was drawn by plotting concentration of the standard on the X-axis versus absorbance on the Y-axis. The amount of carbohydrate present in the tube was calculated from the graph.

Estimation of protein: Protein content was estimated by Lowry’s method [3].

Reagents

A. 2% sodium carbonate in 0.1N NaOH

B. 1% sodium potassium tartarate solution

C. 0.5% copper sulphate solution

D. 48 ml of A, 1 ml of B, 1 ml of C

E. 1 part Folin-Phenol (2N): 1 part water

500 mg of the leaves was weighed and ground well with a pestle and mortar in 5-10 ml of the phosphate buffer. This solution was centrifuged and the supernatant was used for protein estimation. 0.2, 0.4, 0.6, 0.8 and 1 ml of the working standards (Bovine Serum Albumin) were pipetted into a series of test tubes 0.1 ml and 0.2 ml of the sample extract was pipetted in two other test tubes. The volume was made to 1 ml in all the test tubes. The tube with 1 ml of water served as the blank. 5 ml of reagent D was added to each tube including the blank. This was mixed well and allowed to stand for 10 min. Then 0.5 ml of reagent E was added, mixed and incubated at room temperature in the dark for 30 min. Blue colour was developed. The readings were recorded at 660 nm. Standard graph was drawn and the amount of protein in the sample was calculated.

Estimation of chlorophyll: The chlorophyll content in leaves was estimated by the method of Arnon [4].

Reagent is 80% acetone.

Procedure: 1 g of the leaf tissue was weighed and ground with 20 ml of 80% acetone. This solution was centrifuged at 5000 rpm for 5 minutes and the supernatant was transferred to 100 ml volumetric flask. This procedure was repeated until the residue becomes colorless. This was made to 100 ml with 80% acetone. This absorbance was recorded at 645, 663 and 652 nm against the solvent (80% acetone) blank. The amount of chlorophyll present in the extract mg chlorophyll per g tissue was calculated using the following equations.

where

A=Absorbance at specific wavelength

V=Final volume of chlorophyll extract 80% acetone

W=Fresh weight of tissue extracted

Assay of superoxide dismutase (SOD): SOD was assayed according to the method of [5].

Reagents

1. Sodium pyrophosphate buffer (0.025M, pH 8.3)

2. Phenazine methosulphate (PMS) (186 μM)

3. Nitroblue tetrazolium (NBT) (300 μM)

4. NADH (780 μM)

5. Glacial acetic acid

6. n-butanol

7. Potassium phosphate buffer (50 mM, pH 6.4)

Procedure:

Preparation of enzyme extract: The leaves (0.5 g), were ground with 3.0 ml of potassium phosphate buffer, centrifuged at 2000 g for 10 minutes and the supernatants were used for the assay.

Assay:

The assay mixture contained 1.2 ml of sodium pyrophosphate buffer, 0.1 ml of PMS, 0.3 ml of NBT, 0.2 ml of the enzyme preparation and water in a total volume of 2.8 ml. The reaction was initiated by the addition of 0.2 ml of NADH. The mixture was incubated at 30°C for 90 seconds and arrested by the addition of 1.0 ml of glacial acetic acid. The reaction mixture was then shaken with 4.0 ml of n-butanol, allowed to stand for 10 minutes and centrifuged. The intensity of the chromogen in the butanol layer was measured at 560 nm in a spectrophotometer. One unit of enzyme activity is defined as the amount of enzyme that gave 50% inhibition of NBT reduction in one minute.

Assay of catalase (CAT): Catalase activity was assayed following the method [6].

Reagents

1. Phosphate buffer: 0.067M (pH 7.0)

2. Hydrogen peroxide (H₂O₂) (2 mM) in phosphate buffer

Procedure:

Preparation of enzyme extract: 20% homogenate of the leaves was prepared in phosphate buffer. The homogenate was centrifuged and the supernatant was used for the enzyme assay.

Assay: H₂O₂-phosphate buffer (3.0 ml) was taken in an experimental cuvette, followed by the rapid addition of 40 μl of enzyme extract and mixed thoroughly. The time required for a decrease in absorbance by 0.05 units was recorded at 240 nm in a spectrophotometer. The enzyme solution containing H₂O₂-free phosphate buffer served as control. One enzyme unit was calculated as the amount of enzyme required to decrease the absorbance at 240 nm by 0.05 units.

Assay of peroxidase (POD): The method proposed by [7] was adopted for assaying the activity of peroxidase.

Reagents

1. Pyrogallol: 0.05M in 0.1M phosphate buffer (pH 6.5)

2. H₂O₂: 1% in 0.1M phosphate buffer, pH 6.5

Procedure:

Preparation of enzyme extract: 20% homogenate of the leaves was prepared in 0.1M phosphate buffer (pH 6.5) centrifuged at 3000 rpm and the supernatant was used for the assay.

Assay: To 3.0 ml of pyrogallol solution, 0.1 ml of the enzyme extract was added and the spectrophotometer was adjusted to read zero at 430 nm. To the test cuvette, 0.5 ml of H₂O₂ was added and mixed. The change in absorbance was recorded every 30 seconds up to 3 minutes in a spectrophotometer. One unit of peroxidase is defined as the change in absorbance/minute at 430 nm.

The results revealed a drastic decrease in the levels of carbohydrate and protein in cadmium treated plants in both the concentrations compared to control plants, coincides with the massive decrease in barley plant and [8] who found decrease in soluble protein content in plant when grown on various amendments of tannery waste containing heavy metals (Table 1).

Table 1: Effect of cadmium chloride on carbohydrate and protein content on the leaves of Vigna radiate.

The level of chlorophyll a, b was found to be 1.011 ± 0.24, 0.26 ± 0.04 in control plants which has been reduced in 10 and 50 μM cadmium treated plants (Table 2). The decline in chlorophyll content in plants exposed to Cd²⁺ stress is believed to be due to (a) Inhibition of important enzymes, such as protochlorophyllide reductase [9] and δ-aminolevulinic acid dehydratase (ALA- dehydratase) associated with chlorophyll biosynthesis [10] (b) Impairment in the supply of Mg²⁺, Fe²⁺, Zn²⁺, and Mg²⁺ [11]. The decrease in chlorophyll content was also reported in sunflower and in almond [12] (Table 3).

Table 2: Effect of cadmium chloride on chlorophyll content on the leaves of Vigna radiate.

Table 3: Effect of cadmium chloride on superoxide dismutase, catalase, peroxidase activity on the leaves of Vigna radiate.

SOD - Amount that causes 50% reduction in the extent of NBT oxidation.

CAT - Amount of enzyme required decreasing the optical density by 0.05 units.

PEROXIDASE - 1 Micro Molar pyrogallol oxidized per min.

The level of SOD was found to be 18.83 ± 0.24 in control plants, which has been reduced in 10 and 50 μM cadmium treated plants. SOD is a key enzyme in the plant antioxidant defences and is encharged of the dismutation of O₂^- radicals toH₂O₂ and O2 [13] Reduction of SOD activity induced by Cd was reported in wheat [14] and bean plant [15]. The level of Catalase was found to be 283.33 ± 1.99 in control plants, which has been reduced in 10 and 50 μM cadmium treated plants. The decrease of Catalase activity in the presence of Cd ions was also described in pea plant [16]. The level of Peroxidase was found to be 24.69 ± 0.31 in control plants, which has been reduced 10 and 50 μM cadmium treated plants. Peroxidase was down regulated probably by enzyme inhibition by the Cd metal, reported in wheat roots [17]. Hence the present investigation also supports the fact that CdCl₂ can induce a concentration-dependent oxidative stress situation in leaves, characterized by the inhibition of the antioxidant systems.