MATERIALS AND METHODS

Plant Material
The experimental plant used in this investigation was pure strain of common bean (Phaseolas vulgaris L. cv. Nebraska). Seeds were kindly obtained from the Agricultural Research Center in Giza, Egypt.

Experimental Design
For plantation, 25 plastic pots (20 cm) were filled with homogenous pre-sieved garden soil (loamy sand). Surface sterilized common bean seeds were soaked in the pot soil 3.0 cm deep and all pots were watered up to saturation. Seedlings were thinned to three plans per pot, kept in the open garden and irrigated regularly to field capacity until treatments.

Treatments
After two weeks from soaking, the planted pots were randomly subdivided into five equal groups (5 pots each). One group was kept irrigated with pure water and sampled as control. Another two groups were treated with two concentrations (100 and 200 μM) of CdCl₂. The last two groups were subjected to two combinations (100 μM + 100 mM and 200 μM + 100 mM) of CdCl₂ + CaCl₂, respectively.

Sampling and Measurements
After eight weeks from plantation, stem and root lengths, fresh and dry weights, leaf, flower and pod numbers were estimated and recorded.

Statistic
All parameters were statistically analyzed by multiple comparison procedure at p≤0.05 using t-test and mean separation by Least Significant Difference (LSD) (Steel and Torrie, 1980).

Estimation of Photosynthetic Pigments
The contents (μg g^-1 dry weight of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids were estimated in the fresh leaves by the method of Lichtenthaler and Wellburn (1983).

Protein Electrophoresis
Extraction of Total Protein
Total protein extracts were prepared by extracting appropriate weight from the frozen plant material with 0.125 M Tris/borate, pH 8.9. All the obtained extracts were kept at 4 °C for 24 h and centrifuged at 10000 rpm for 20 min. The supernatants were used for electrophoresis.

Gel Electrophoresis
SDS-PAGE was carried out with gel slabs according to the method of Laemmili (1970). Protein subunit bands were stained with Coomassie blue R-250 by standard techniques. The gel was scanned using Gel pro- Analyzer ver. 3.3 (Media Cypermetics 93-97).

Histological Examination
Eight-week-old common bean plant roots were used. Plants were cultivated under similar conditions of Cd toxicity and Ca effect assay. Root specimens were fixed in formalin: acetic acid: ethanol (FAE). Tissues were dehydrated in n-butyl alcohol, infiltrated and embedded in pure paraffin wax (m.p. = 56-58 °C) as described by Johansen (1940). A rotary microtome was used to prepare serial sections (10 μ), which were then stained with safranin and light green or hematoxilin. Stained sections were examined and photographed with Zeiss Microscope.

RESULTS

Growth Parameters
Cd Phytotoxicity caused noticeable growth retardation in most of the growth parameters of common bean (Phaseolas vulgaris L.) plants. These growth parameters were significantly reduced by both 100 and 200 μM CdCl₂ (Table 1). The stem length, stem fresh weight, stem dry weight and number of pods per plant were significantly reduced by both concentrations (100 and 200 μM) of CdCl₂; while, the number of leaves and number of flowers per plant were reduced only by the high concentration (200 μM) of CdCl₂. The root growth parameters were not significantly responding to Cd toxicity. The addition of CaCl₂ (100 μM) to Cd-stressed (100 μM CdCl₂) common bean plants improved the stem fresh weight, root length, number of flowers and number of pods per plant.

Photosynthetic Pigments
As shown in Table 3, the contents of chlorophyll a, chlorophyll b and total chlorophyll were reduced in foliage leaves of common bean plants in response to the applied concentrations (100 and 200 μM) of CdCl₂; while, carotenoids were higher in Cd-treated plants. The addition of CaCl₂ increased the content of chlorophyll a and total chlorophyll, but not chlorophyll b.

Protein Electrophoresis
Cd stress induced both qualitative and quantitative changes in SDS-PAGE protein profile of common bean plants (Fig. 1, Table 2). The total number of protein band was reduced from 27 in untreated (controlled) plants to 25 and 23 bands in plants treated with 100 and 200 μM CdCl₂, respectively. However, addition of CaCl₂ (100 mM) did not correct the changes in the number of protein bands caused by Cd stress. The synthesis of high molecular weight proteins (116 and 85.54 kDa) was completely inhibited by 200 μM CdCl₂; while, the synthesis of low molecular weight protein (26.11 kDa) was totally blocked by both CdCl₂ treatments. The addition of 100 mM CaCl₂ restored the synthesis of a 28.57 kDa protein. The synthesis of a molecular weight polypeptide of 101 kDa was induced by the high concentration of CdCl₂.

Table 1:	Mean vegetative and reproductive growth parameters of common bean (Phaseolas vulgaris L. cv. Nebraska) plants treated with CdCl2 (100 and 200 μM) and CdCl2 + CaCl2 (100 or 200 μM + 100 mM), respectively
C: Controlled plants, T1, T2: Plants treated with 100 and 200 UM CdCl2, respectively, T3: Plants treated with 100 uM CdCl2+100 mM CaCl2, T4 = Plants treated with 200 uM CdCl2+100 mM CaCl2

Table 2:	Comparative analysis of relative concentration, molecular weight (M.wt.) and rate of mobility (Rm) of SDS-PAE protein profile of common bean (Phaseolas vulgaris L. cv. Nebraska) plants treated with CdCl2 or CdCl2 + CaCl2
T1: Untreated (control) plants, T2 and T3: Plants treated with 100 and 200 μM CdCl2, T4 and T5: Plants treated with 100 CdCl2 +100 mM CaCl2 and 200 μM CdCl2 + 100 mM CaCl2; respectively

Table 3:	Photosynthetic pigments (μg g-1) in foliage leaves of common bean (Phaseolas vulgaris L.) C, untreated (Controlled) plants treated with CdCl2 or CdCl2 + CaCl2
C: Untreated (control) plants, T1 and T2: Plants treated with 100 and 200 μM CdCl2, T3 = Plants treated with 100 μM CdCl2 + 100 mM CaCl2, T4: Plants treated with 200 μM CdCl2 +100 mM CaCl2

Histological Staining
Thin sections from common bean root (Fig. 2) indicated progressive collapsing and disruption of the epidermal and cortical tissue layers of the root tissues and browning of tissues followed by root cell death caused by CdCl₂ treatments (Fig. 2B, C) compared to the root of untreated plants (Fig. 2A). The addition of CaCl₂ relatively prevented the brown coloration and collapsing of tissues caused by the low and high concentrations of CdCl₂ (Fig. 2D, E).

Fig. 1:	Electrophotograph of SDS-PAGE of total proteins of common bean (Phaseolas vulgaris L. cv. Nebraska) plants. 1, untreated (controlled) plants (track 1); 2 and 3, plants treated with 100 and 200 μM CdCl2 (tracks 2 and 3); 4 and 5, plants treated with 100 and 200 μM CdCl2 + 100 mM CaCl2 (tracks 4 and 5); M, molecular weight markers used on polyacrylamide gel

Fig. 2:	Effect of CdCl2 on the epidermal and cortical tissues of common bean (Phaseolas vulgaris L.) plant roots. (A) untreated plants, (B and C) plants treated with 100 and 200 μM CdCl2, respectively, (D) plants treated with 100 μM CdCl2 + 100 mM CaCl2 and (E) Plants treated with 200 μM CdCl2 + 100 mM CaCl2, respectively

DISCUSSION

In common bean plants, the results of this investigation reported on a variety of phytotoxic effects of Cd to plant growth, chlorophylls, protein synthesis and root tissue damages as well. Consistent findings reported that free Cd and other heavy metal ions often reduce cellular activities for a variety of reasons; for example, by generating oxidative stress and inhibition of enzyme reactions. Suzuki (2005) reported on plants incubated for two weeks in a sublethal level (200 μM) of Cd, root cells survived but with irregular thickening of cell walls and enlarged and unusually formed cells. Although it is not clear which mechanism caused those growth changes in root cells, Cd may have altered some metabolic pathways i.e., cell division block through the lack of Glutathione (GSH) integrity in plant roots (Vernoux et al., 2000), or Cd affected directly GSH metabolism (Xiang and Oliver, 1998).

In this report, it was showed that Ca reduced the toxic effects of Cd in common bean plants. Relevant results reported that Ca reduced the uptake, accumulation and toxic effects of Cd in Arabidopsis (Suzuki, 2005) and radish (Rivetta et al., 1997). Cd tolerance increased in tobacco in the presence of Ca, that probably due to the active exclusion of toxic Cd by the formation and excretion of Cd/Ca containing crystals through the head cells of trichomes (Choi et al., 2001). It was also previously reported that the accumulation of Cd on seed germination of radish is reduced by high concentration of Ca (Rivetta et al., 1997).

Several mechanisms for Ca alleviation of mineral toxicity have been adopted. A proposed mechanism is the displacement of cell-surface toxic cations by Ca. Since plasma membrane surfaces are negatively charged, high level of Ca⁺² would reduce cell-surface negativity and alleviate the harmfulness of cationic toxicants (Kinraide, 1898). The other proposed mechanism is the uptake of Cd through calcium channels blockers, diltiazem, verapmil, nifedipine and nitrendipine (Blazka and Shaikh, 1991). It is evident that a large number of carrier proteins are involved in the transport of Cd (Maser et al., 2001). As a major defense mechanism, the inactivation of these metal ions could be accomplished by complexing them with phytochelatins such as metalothionin and cysteine-rich proteins (Di Toppi and Gabbrielli, 1999; Song et al., 2004).

In conclusion, Ca ion could be applied in alleviating Cd Phytotoxicity in common bean plants in moderately Cd-contaminated soils^. Such treatments would be worked out if the Cd concentration outside could be kept below a critical threshold level (Haag-kerwer et al., 1999). Further investigations would focus on elucidating the precise role of Ca in decreased Cd influx into plants.