Abstract: Seed germination and early growth of bean (Phaseolus vulgaris) seedlings were stimulated by irrigation with sewage water. The stimulation of growth was accompanied by induction in synthesis of pigments, carbohydrates, nucleic acids, nitrogenous compounds, except amino acids-N decreased. The activity of amylase, invertase and protease enzymes increased may be due to the presence of some mineral ions which act as activators for enzymes. Treatment of sewage water by precipitation, rice residue or EDTA decreased its enhancing effect. Protein banding patterns of the produced seeds indicated that the appearance of new bands having high molecular weights was the marked feature in sewage water irrigated plants. The concentration of Zn, Cu, Cd and Pb increased greatly in the different plant organs in response to sewage application. Treating the sewage by precipitation, rice residue, or EDTA reduced their accumulation, particularly with precipitation treatment.
INTRODUCTION
Sewage water and sewage sludge is used nowadays to improve physical, chemical properties and fertility index of soil in order to increase production per unit area. Pollution with ions of heavy metals result from excessive and/or unplanned uses of sewage water. In small quantities, certain heavy metals are nutritionally essential for a healthy life e.g., Fe, Cu, Mg and Zn. Increased loading of heavy metal ions in water and soil produce increases in mutations or cell death in plants and increases in human health hazards as the heavy metals enter and concentrated in the food chain through uptake by plants and ingestion by animals (Sancherz et al., 1999).
The residual effect of sewage or sewage sludge applications increased significantly plant height, fresh and dry weight, grain yield, number of pods/plant and seed yield (Lutrac et al., 1982), as well as leaf area and leaf biomass (Brown, 1981). Harangozo et al. (1984) added that pretreatment of vicia faba (broad bean) seeds with sewage water led to increase length of both root and shoot. Sewage sludge increased chlorophyll content and oxygen evolution (Drewa et al., 1993), as well as total soluble sugars and sucrose content (Mellberye et al., 1982). Nitrogen content also increased by sewage sludge application in wheat straw (Roszyk et al., 1989) and in pea and cotton plants (Lewis et al., 1992). Nitrate-nitrogen content decreased in plants irrigated with sewage (Belyuchenko and Dronov, 1988). Fresquez et al. (1990) reported that most nutrients including plant nitrogen were linearly increased as a result of sludge amendment. However, Ito et al. (1991) observed that sewage sludge application at the rate of 60 t ha-1, led to inhibition of growth due to rapid mineralization of nitrogen, which enhances accumulation of toxic ammonia to growth. The activity of proteinase and peroxidase increased in potato stem and root, respectively in response to sewage water application (Chakrbarti and Chakrabarti, 1987).
Chelation process is simply defined as process by which a molecule encircles and binds (attaches) to the metal and removes it from the medium. It occurs mainly through the induction of metal binding peptides and proteins such as metallothioneins and polyhistidindes (Mejre and Bulow, 2001). EDTA is one of the oldest chelating agents, Namasivayam and Ranganthan (1998) reported that Citrate and EDTA considerably decreased the adsorption of Ni2+, while Cd2+ adsorption decreased significantly in the presence of acetate and citrate. Azenha et al. (1995) found that the exposure of P. syringae cells to 100 μM copper alone led to its death, suggesting that copper was responsible for cell death. EDTA significantly reduced both the amount of copper bound to the cells and cell death, indicating that, not only strong chelating agent but also weak and moderate copper ligands can effectively antagonize copper toxicity. Residues such as molasses, blood mealand silage effluents, containing various aliphatic carboxylic acids, sugar acids and amino acids or other precursor compounds have a potential to serve as extractants. Xiong (1994) reported that Cd adsorption significantly increased when the soil had previously been incubated with the rice straw and a milvetch (Astralagus shoot), the results of their study indicated that the pH of the treated soil samples rose after incubation, changes in pH were hence considered responsible for increasing Cd adsorption by plant material incubated soil samples. Apple Residues (AR) were used for removal of the heavy metals Cu, Pb and Cd with higher selectivity for Pb than Cu and Cd. The presence of organic ligands decreased the capacity for Cu removal due to metal-ligand complex formation. AR may be used as effective and inexpensive adsorbent for metal removal from aqueous solution (Sung et al., 1998).
The present research aims to study the effect of sewage water usage for irrigation of crops e.g., bean (Phaseolus vulgaris). The study included germination, seedling and fruiting stages with respect to germination percentage, growth, some enzyme activities, Hill reaction and some metabolites content, as well as the protein banding patterns of the produced seeds, as indication of gene expression, as affected by heavy metals in the sewage water. The concentrations of Zn, Cu, Cd and Pb were measured in the different plant organs.
MATERIALS AND METHODS
Sewage water was obtained from Arab Abou Saed region, south of Helwan. The experiment was carried out to study the effect of sewage water usage on germination and growth of bean (Phaseolus vulgaris) as one of the plants cultivated in the sewage-polluted area. Its effect on photosynthetic pigments, carbohydrates, soluble protein, DNA, RNA, nitrogenous compounds and some enzyme activities were also studied. Some treatments e.g., precipitation, rice residue and EDTA were applied to reduce the accumulation of heavy metals in sewage water irrigated plants. 1N NaOH was added for precipitation of heavy metals, then filtered through cotton plug and neutralized before using for irrigation. Rice residue was used as 0.5 kg/kg soil and EDTA was used at 50 mM concentration. Seeds of bean were obtained from the Agricultural Research Center, Ministry of Agricultural, Giza, Egypt.
For enzyme assay, plant material was prepared by macerating the tissues with a chilled pestle and mortar at 0-4°C. The tissue homogenate was centrifuged at 10 000 g for 20 min and the supernatant obtained was used directly for determining enzyme activity. For assaying the activity of α- and α-amylases, 3,5-dinitrosalicylic acid reagent was used according to Bergmeyer (1974). Acid-invertase enzyme was assayed by the method of Malik and Singh (1984). Protease activity was assayed according to the method described by Bergmeyer (1974). Fresh leaves were extracted in 70% ethanol and completed to a known volume with distilled water and used for estimation of total sugars using anthrone reagent (Umbriet et al., 1959), while the reducing sugars were determined by using Nelson reagent according to Naguib (1969). Photosynthetic pigments were estimated in 85% acetone extracted leaves according to Metzener et al. (1965). For isolation of chloroplasts, according to the method of Aronoff (1946) and Osman et al. (1982), fresh leaves were blended in cold buffer with 0.4 M sucrose, (pH 7.8), 3 mM MgCl2, 4 mM sodium ascorbate and 0.1% bovine serum albumin. The suspension was centrifuged at 4°C (1 min at 800 g). The pellet was resuspended in the isolation buffer and centrifuged for 5 min at 300 g and the supernatant was then centrifuged for 10 min at 1000 g. Chloroplasts (residue) were resuspended in the buffer solution. Hill reaction of the isolated chloroplasts was measured by using potassium ferricyanide as electron acceptor. Borate buffer (pH 8.0) extract of dry leaves was used according to Naguib (1969) for determination of soluble nitrogenous compounds e.g., nitrate-N, amino acid-N, peptide-N and total soluble-N. Total nitrogen was measured by digesting the dry leaves in 50% sulphoric acid and 35% perchloric acid and its ammonia content was estimated using Borthelot reaction which carried out according to Chaney and Marbach (1962). DNA was measured according to Dische and Schwartz (1973) by using diphenylamine reagent. RNA was determined using the method adopted by Ashwell (1957) using orcinol reagent. Protein banding patterns were examined in the Central Laboratory of Horticulture Research Center, Giza, Egypt. Separation of proteins was performed using Sodium Dodicyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE).
Statistical analysis was carried out according to Snedecor and Cochran (1980) using analysis of variance and the significance was determined using LSD values at p = 0.05 and 0.01.
RESULTS AND DISCUSSION
Sewage water application increased germination percentage of bean seeds to become 98% compared with control seeds (92.5%). The increased germination percentage because of sewage application was associated with a stimulating effect on the hydrolytic enzymes α- and β-amylases and proteases. Treatment of sewage water by precipitation, rice residue or EDTA led to a significant decrease in its stimulatory effect on germination percentage and enzymatic activities (Table 1). Application of sewage water significantly increased growth criteria e.g., shoot and root lengths, fresh and dry masses of bean seedlings, as well as the yield criteria e.g., number of pods/plant, length of pods, fresh and dry weights of pods. Treatment of sewage water by precipitation, rice residue or EDTA reduced its stimulatory effect. The beneficial effects of sewage application may be due to the greater capacity of sewage to supply nutrients to the plant and to improve soil properties (Heckman et al., 1986). These nutrients may activate the hydrolytic enzymes during germination, which in turn increase the amount of the hydrolyzates e.g., glucose and amino acids, which are required for growth of embryo axes (Zeid and Shedeed, 2006). The obtained results also support the suggestion of Staniforth and Smith (1991) who attributed the increased crop yield as a result of increasing rate of sewage application to the available nutrients supplied particularly nitrogen, which is reflected in the increased total nitrogen content of the crop.
Application of sewage water positively affected synthesis of photosynthetic pigments and photosynthetic activity (Hill reaction). Treatment of sewage water with precipitation, rice residue or EDTA reduced this increment (Table 2). The stimulatory effect of sewage water on pigments content could be attributed to the fact that sewage water enhances the rate of biosynthesis of chlorophyll a and b. The increased content of chlorophyll was in parallel with the enhancement of plant growth i.e., there is an intimate relationship between growth and chlorophyll content. El-Maghraby and Gomaa (1992) reported that sewage water application increased number of green leaves and leaf area per plants, or it may be increase both of macro and micronutrients elements in soil, which is essential for the plant growth and photosynthetic pigments.
Total soluble sugars, polysaccharide and total carbohydrates content were higher in sewage water irrigated plants compared with controls and decreased by treating the sewage by precipitation, rice residue, or EDTA (Table 2). The effect of precipitation treatment was more obvious. The increased content of carbohydrates in sewage water irrigated plants may be due to the presence of some mineral ions e.g., Mn and Cu that stimulate the two photosystems. Mn+2 is required for PSII (O2 evolving system) and there is also a direct interaction between copper and ferredoxin on the reducing site of PSI. Cu++ stimulate the rate of overall electron transfer from water to NADP (Marschner, 1986).
DNA and RNA were significantly increased in sewage water irrigated plants (Table 3), may be due to the enrichment of sewage water with heavy metals such as Zn, which is a component of carbonic anhydrase, RNA polymerase and DNA polymerases. Treatment of sewage water with precipitation, rice residue or EDTA reduced its stimulatory effect on germination, growth, photosynthetic pigments, Hill reaction, carbohydrates, nucleic acids and hydrolytic enzymes activity due to the removal or chelation of heavy metals present in sewage water. Precipitation treatment was more effective. Kasan and Baecker (1989), Azenha et al. (1995) and Namasivayam and Ranganthan (1998) reported that EDTA treatment was effective as a chelator for sludge bound metal-ions.
| Table 1: | Effect of sewage water and treated sewage water on germination, enzyme activities during germination μg g-1 f.m. h-1), growth of seedlings (15-day-old) and yield (90-day-old) of bean |
| Pptn: Precipitation | |
| Table 2: | Effect of sewage water and treated sewage water on the leaf content of photosynthetic pigments, Hill reaction (μM [ferricyanide] g-1 chlorophyll s-1), carbohydrate content (mg g-1 dm) and enzyme activities (μg g-1 fm h-1) in leaves of bean seedlings (15-day-old) |
| Pptn: Precipitation | |
Soluble nitrogenous compounds e.g., peptides, total soluble nitrogen, as well as total nitrogen increased in response to sewage water, while the nitrate-N and amino acids nitrogen content decreased (Table 3). However precipitation, rice residue or EDTA treatments reduced the accumulation of peptides, total soluble nitrogen, as well as total nitrogen, while the nitrate-N and amino acids nitrogen content increased, particularly with precipitation treatment. The reduced content of amino acids by sewage water application may be attributed to the stimulation of nitrate reduction process and consequently the ammination process to form protein (Ito et al., 1991). Marschner (1986) reported that Mn at certain levels plays a direct role in oxidation-reduction reaction and play an essential role in protein biosynthesis. Boardmen (1975) stated that the role of Mn in N03-N reduction step is expected as a kind of indirect relationship between Mn toxicity and N-assimilation by plants.
Activities of α- and β-amylases, invertase (Table 2) and protease (Table 3) also increased in leaves of sewage water irrigated plants. This activation may be due to the uptake of some mineral ions e.g., Mn, Zn and Fe, which stimulate these enzymes and consequently increase the cellular content of soluble sugars, reducing sugars and peptides, as indicated in Table 2 and 3. On the other hand, application of precipitation, rice residue or EDTA decreased this activation, but still higher than that of the controls.
Concerning the protein banding patterns, which indicated the effect of heavy metals on gene expression, the appearance of protein bands having high molecular weights was the marked feature in the sewage water irrigated plants (Table 4 and Fig. 1). In general, the seeds of control plants showed 24 bands with molecular weights ranged between 195.4 and 5.3 KDa. Seeds produced from plants irrigated with sewage water exhibited a new 9 polypeptides with molecular weight 210, 140, 107, 84.9, 69.11, 45.4, 53.5, 22.4 and 7.3 KDa.
| Table 3: | Effect of sewage water and treated sewage water on the leaf content of nitrogenous compounds (mg g-1dm), DNA and RNA (mg g-1 fm) and protease activity (μg g-1 fm h-1) in leaves of bean seedlings (15-day-old) |
| Pptn: Precipitation | |
| Table 4: | Protein profile of the produced seeds by bean plants in response to sewage and treated sewage water irrigation |
| Pptn: Precipitation | |
| Table 5: | Concentration of Cu, Zn, Pb and Cd (μg g-1 dm) in the different plant organs of bean as affected by sewage and treated sewage water |
| Pptn: Precipitation | |
| Fig. 1: | Electrophotograph of protein banding patterns of bean seeds produced from plants irrigated with: 1- control water, 2- sewage water, 3- sewage water treated with precipitation, 4- sewage water treated with rice residue and 5- sewage water treated with EDTA |
At the same time, 9 bands with M.Wt. of 195.4, 124, 118, 80, 98.11, 55.5, 50.4, 20.1 and 5.3 KDa disappeared, in comparison with the control. Seeds produced from plants irrigated with sewage water after precipitation treatment showed a more or less similar protein bands to that of the control plants, with only new three bands having molecular weights of 84.6, 45.1 and 22.1 KDa appeared. Seeds produced from plants irrigated with sewage water treated with rice residue, showed 23 bands and the mobilities ranged between 0.066 and 0.978. Major variation was indicated by the appearance of protein bands similar to that of sewage water irrigated plants, but have lower molecular weights e.g., 203, 128.5, 84.2, 69.03, 48.4 and 38.11 KDa. Only one protein band with mobility of 0.991 disappeared. EDTA treated sewage water resulted in seeds having only 23 bands. Three new bands with lower molecular weights (200, 62.54 and 28.43 KDa) than that of sewage irrigated plants were observed and also one band of mobility 0.991 was completely missed.
The appearance or disappearance of new bands was attributed either to alternation in the structural genes, or changes in the expression of regulatory genes involved in regulating these genes due to mutagenic effect of heavy metals present in sewage water. Mutational events occurring in the regulatory genes may lead to inhibition or constitutive expression of concerned genes and this will result in the disappearance of some bands or changes in some band intensities i.e., heavy metals present in sewage water result in an increase in the transcription of a number of stress-induced genes and lead to the accumulation of their polypeptides. Giordani et al. (2000) observed an accumulation of transcripts after exposure to trace metals such as copper and cadmium. The biological effects of heavy metals have been investigated for many years by many authors.
Irrigation of bean plants with sewage water led to accumulation of Zn, Cu, Pb and Cd in all plant organs (Table 5). Precipitation, rice residue, or EDTA treatments reduced this accumulation, but still more than plants irrigated with control water. Precipitation treatment was more effective. Antonovics et al. (1971) reported that one of the major concerns with the utilization of sewage water and sludge in agricultural crop production is the metal enrichment of plant tissue. Dowdy and Larson (1975) stated that the accumulation of metals in vegetable tissue was higher than in the edible fruit, root and tuber tissues following sewage application. Accumulation of Zn, Cu, Cd and Pb in plants irrigated with sewage water was also, observed by Maclean et al. (1987) and Berton et al. (1989).
CONCLUSIONS
In fact, the sewage water application increased germination percentage, growth, metabolic activities and the produced yield of bean, but the concentration of heavy metals, which have hazardous effects on human and animal health, was higher than permissible for health. Electrophoretic protein banding pattern, which is a mirror image for transcription events occurring during gene expression, indicated the appearance of new bands having higher molecular weights and disappearance of others. Finally, it could be suggested that sewage water must not be used without treatment to reduce the accumulation of heavy metals in the edible plants.