EFFECT OF SEQUENTIAL CONCENTRATIONS OF ZINC AND ITS COMBINATION WITH CALCIUM OR GLUTATHIONE ON THE GROWTH, WATER RELATIONS AND ANATOMY OF ROOTS, STEMS AND LEAVES OF PHASEOLUS VULGARIS CV. CONTENDER

Zinc (Zn) is one of the eight essential micronutrients. It is needed by plants in small amounts, but yet crucial to plant development. A solution culture experiment was conducted to study the variation in growth, water relations and anatomy of roots, stems and leaves of Phaseolus vulgaris cv. contender treated with 1, 200, 400, 600, 800, 1000and 1200 mMZnSO4. Maximum significant deplete in parameters of growth (Length of root and shoot; fresh and dry weights, relative growth rate; N of leaves and leaf area), stomatal index and rate of transpiration was observed with, 200, 400, 600, 800, 1000and 1200 mM ZnSO 4 . These effects were improved by the addition of Ca 2+ than the addition of glutathione at 10 mM. Moreover, there were a significant increase at low concentration (1mM) treatment. Width of root, width of cortex and width of vascular bundles were increase with increasing Zn concentrations either alone or in combination with glutathione >Ca (NO 3 ) 2 . For stem, the N of vascular bundles were decreased with increase in Zn concentration alone and with Ca (NO 3 ) 2 , meanwhile increase with glutathione. Width of cortex and N of its rows were decrease with increase Zn concentrations. For leaves, the thickness of leaf blade, mid rib and vascular bundle were increase with increase the Zn concentrations alone, On other hands, they records a significant decrease in combination of Zn with Ca(NO 3 ) 2 or glutathione . In general, an increase in total uptake of zinc with increasing the concentration of Zn in all treatments. However, Ca (NO3)2 decrease these amounts than glutathione.


Introduction
The term "heavy metal" commonly refers to metals with a specific weight in excess of 5 g / cm or anatomical number in excess of 20. Such properties are our significance for biological effects, yet the heavy metals contain essential nutrients, beneficial elements and elements that are not known to be active in humans at the present stage of science. At relatively low levels, all of them become harmful. Yet toxicity is not an exclusive aspect of heavy metals listed elements. The heavy metals are therefore a rather heterogeneous group of elements which differ greatly in their chemical properties and biological functions. The word "heavy metal" is therefore debunked (Nieboer and Richardson 1980). But as Tiller (Tiller 1989) pointed out "heavy metal may be a useful umbrella term for metals classed as environmental pollutants". Among the myriad of heavy metals zinc occupies the prominent position, since it plays a vital role in the growth and development of plants. Zinc, one of the essential micronutrients and an important constituent of a number of enzymes and proteins, is required only in small quantities by plants. However, plant development is crucial, as it plays a significant role in a wide range of processes. The normal range for zinc in plant tissue is between 15-60 ppm and between 0.10-2.0 ppm in the growing medium. Zinc deficiency or toxicity is not common; however, both negatively impact crop growth and performance. Any deficiency or toxicity must be addressed prior to irreversible crop damage .
Zinc release to the atmosphere may be correlated with biotic and natural atmospheric processes, with a ratio of Zn emissions from human activities to those from natural causes exceeding 20. (Friedland 1990). Human activities that release Zn to the atmosphere include fossil fuel combustion and the use of sewage sludge, manure and lime. Many crops may suffer from Zn toxicity in polluted and acidic soils, and species with high Zn uptake potential, such as spinach and beet, may be more prone to its abundance. (Chaney 1993 Calcium is considered to have a positive influence on plant growth and to enhance heavy metal toxicity. Hagemeyer, 1999). In addition, Ca was found to decrease the content of Cd, Cu, Mn and Zn in plant roots and/or shoots (Kawasaki and Moritsugu, 1987; Salehet al., 1999). In order to handle different types of metals, plants have protection techniques linked to cellular free metal content (e.g., metal exclusion, cell wall binding, chelation and sequestration) on one side. (Hall, 2002) However, on the other hand, control of cellular responses (e.g. repair of stress-damaged proteins, antioxidant protection). (Hall, 2002). The synthesis of specific chelators and subsequent sequestration of metal complexes is of major importance to limit free metal concentrations. Glutathione (GSH) is a key component of such metal scavenging due to the high metal affinity with its thiol (-SH) group and as a phytochelatin precursor (PC). In addition to metal homeostasis, plants have a well-equipped antioxidant defense system to deal with the metalimposed oxidative challenge (Jozefczaket.al. 2012).
Phaseolus vulgaris, also referred to as the common bean, Gentry, Howard Scott (1969) green bean and French bean, among other names, is a herbaceous annual plant grown worldwide for its edible dry seeds or unripe fruit (both commonly called beans). The main categories of common beans, on the basis of use, are dry beans (seeds harvested at complete maturity), The common bean grows well on large variable soils with pH ranging from 4 to 9. It grows better on well-drained, Http://www.granthaalayah.com ©International Journal of Research -GRANTHAALAYAH [3] sandy loam, silt loam or clay loam soils, rich in organic matter content. Dry beans production (theoretically only Phaseolus species) was about 23 million ton in 2012, cultivated on 29 million ha (FAO, 2013)In recent years, consumption of legumes particularly dry beans ( Phaseolus vulgaris L.) has increased in some West European countries and the United States. This is due to an increased realization of consumers about the nutritional characteristics in foods.
The goal of this study was to investigate the effects of high nutrient solution concentrations of Zn on growth, water relationships, Zn content and anatomy characteristics composition of different parts of the plant bean model (Phaseolus vulgaris L.). The objective was also to determine the role of calcium and glutathione in improving zinc toxicity in plants of Phaseolus vulgaris. Throughout the experimental period, various growth parameters, stomatal index, stomatal area, rate of transpiration, content of zinc in root and shoot were determined. In addition, the changes in the internal structure of root, shoot and leaves were determined.

Time Course Experiment
Data from the different groups of seedlings were statistically analyzed and comparison among means was carried out using Statgraphic Ver. 4.2, Display (one-tailed ANOVA), as described by Snedecor and Cochran 1980).

Growth Parameters
The plant heights from the root system intersection to the stem's growing tip were measured and at the end of the experiment (14-day old) root length was determined. The fresh weights and dry weights of the shoots and roots were obtained using an electronic balance. As well as number of leaves and leaf area were determined. Http://www.granthaalayah.com ©International Journal of Research -GRANTHAALAYAH [4] Relative Growth Rate (RGR) Relative growth rate (RGR) was calculated according to (Hofmann and Poorter 2002) formula: Where W1 and W2 are the dry masses at 7 and 14 -day harvest T1 and T2 respectively on the basis that growth was exponential during this growth period.

Rate of Transpiration
The rate of transpiration was estimated gravimetrically from the decrease in the weight of the whole plant and culture solution on the basis of root fresh mass as mg g-1 fresh mass h -1 . (Youniset al., 1992).
Rate of transpiration = W1 -W2 ______________________________________ = mg/g -1 F. Wt root F. wt of root x time of experiment in hours (48 hr) W1 = weight of plant at the beginning of experiment W2 = weight of plant at the end of experiment

Determination of the Stomatal Index
The stomatal number (stomatal density) is called the total number of stomata per square millimeter of epidermis. According to the stomatal index, the percentage proportional to the ultimate divisions of the epidermis of a leaf that has been converted into stomata (Weyers and Meidner, 1990):

Stomatal Index= Stomatal density x100
Stomatal density +density of epidermal cells S SI = _________________ x 100 S +I Where SI = Stomatal index, S = number of stomata per unit area and E = number of ordinary epidermal cells in the same unit area.

Procedure
Pieces of the leaf between the margin or midrib were cleaned and mounted, and the lower surface was examined using a 4 mm objective microscope and an eyepiece with a 5 mm square micrometer disk. The numbers of the epidermal cells and the stomata within a square grid were counted, a cell being counted if at least half of its area is within the grid. The index of stomas was calculated for both surfaces of the leaf.

Chemical Analysis
The plants were harvested at the age of 14, shoots and roots, and the roots were washed with deionized water and the samples were dried for chemical analysis at 80oC in an oven for 48 h. Then dry shoots and roots were weighed and grounded. Plant samples (0.5 g) were digested with Http://www.granthaalayah.com ©International Journal of Research -GRANTHAALAYAH [5] concentrated HNO3 and H2O2 (Jackson, 1958; Han et al., 2004). The digested solution was filtered and then analyzed for Zn atomic absorption spectrophotometry (ICP-AES-Liberty series II) (Han and Banin 1997). Calculated as mM/100gm Dry weight.

Anatomical Preparation
For anatomical investigation, samples from plants were taken after ending the experiment (about 14-days-old). Stem sample were taken from the first internode after the 1 st foliage leaf. Leaf samples were taken from 1 st trifoliate leaf. Root samples were taken 5 cm away from the point of attachment of root and shoot. Plant material was fixed in FAA ( Formalin , acetic acid and alcohol : 1:1:1 ) dehydrated , paraffin embedded, ultramicrotomed and subjected to safranin (0.1%)fast green (0.2%) staining for further observation (Sass 1958).In these sections , thickness of section , number of cortical cells (raws) , width of cortical cells , number of vascular bundels in root and stem were determined . In leaf sections, the blade thickness, Midrib thickness and Width of vascular bundle were determined using linear micrometr. (Shukry 1986).

Results and Dissection
Zinc (Zn) is an essential component of thousands of proteins in plants, although it is toxic in excess. Zinc toxicity in crops is far less widespread than Zn deficiency. However, Zn toxicity occurs in soils contaminated by mining and smelting activities, in agricultural soils treated with sewage sludge, and in urban and peri-urban soils enriched by anthropogenic inputs of Zn, especially in low-pH soils (Chaney 1993  Http://www.granthaalayah.com ©International Journal of Research -GRANTHAALAYAH [6] the abscisic acid content in the root and in the leaves increased significantly. The rise in leaf abscisic acid was correlated with the root material .

Shaukat, et.al (1999)
suggested that shooting heavy metal concentrations lead to high phenolic compounds that could be responsible for germination and growth inhibition. Phenolic acids have been shown to exert dramatic effect on membrane permeability and membrane electrical potentials. Zn concentrations of 100-400 µg g-1(soil d.m.) cause significant decrease in root and shoot growth parameters at different developmental stages of Artemisia annua plants and the biomass decline and inhibition of cell elongation and division (Khudsar et al., 2004) Zinc In Zn-( Table 2), accumulation in plant shoots and roots increased significantly (P < 0.05) either alone or in incompatibility with Ca +2 or glutathione treated groups with increased concentration of applied metal solution. There were a negative correlations between Zn accumulation in shoots and roots to RWC, plant fresh and dry weight and plant height (Fig. 1).
Meanwhile , there were a positive correlations with S/R ratio , this may indicate that , the root is more sensitive to Zn than shoot. Sresty and MadhavaRao )1999) based on transmission electron microscopy concluded that radicle elongation was more adversely affected than the plumule extension. The major change was seen in the nucleus of the root tip cells due to zinc toxicity. The chromatin material was highly condensed and some of the cortical cells showed disruption and dilation of nuclear membrane in presence of 7.5 mM zinc. The cytoplasm became structureless, disintegration of cell organelles and the development of vacuoles were also observed. Http://www.granthaalayah.com ©International Journal of Research -GRANTHAALAYAH [7] stomatal deformation, reduction in frequency of normal stomata in of Phaseolus vulgaris L. cv. Limburgsvroege were sown in peat moss supplemented with ZnSO4 (600 mg kg).
Glutathione combination can mitigate the plant's toxic effect of Zn. In particular, when taken in excessive amounts, all metals can contribute to toxicity and oxidative stress, which poses a serious threat to the environment. Plants have defensive strategies in which glutathione (GSH; π-glu-cysgly) plays a central role as a chelating agent, antioxidant and signaling element in order to cope with different types of metals. This analysis therefore emphasizes GSH's role in: (1) (Kinraide,1998(Kinraide, , 1999. Mechanism III is the residual alleviation beyond Mechanisms I and II. It is a heterogeneous suite of mechanisms that may entail interactions between Ca 2+ and the toxicant at the PM surface

Effect of Zinc Treatment on Zn Concentration in P. Vulgaris
The effect of zinc treatment on its come into roots and shoots of P. vulgaris are presented in table 2. The results were highly significant at all levels of Zinc in the solution culture influenced it's concentration in P. vulgaris. It was observed that when the zinc levels in solution increased its concentrations in roots were also increased. Zinc was accumulated in roots and shoots. The average Zn concentration ranged from 0.416 to 35.91mM in roots in treatment with Zn alone, and from 0.60 to 33.00mM in combination of Zn with Ca (NO3)2 and from 0.69 to 61.63 in combination with glutathione. A gradual increase of zinc concentration was observed with the increasing Zn levels. In shoots Zn concentration was also found to increase with increasing Zn levels. The Zn concentration in P.vulgaris shoots was found lowest than in the roots. Roots accumulate more Zn than shoots. In general, total uptake of Zn decreased in supplementation of ZnSO4 with Ca(NO3)2 than with glutathione .

Effect of Zn Accumulation on Plant Internal Structure
Following the results in Table (3) and images (2), there was an increase in the root thickness. This coincided with an increase in the density of the vascular area and vascular bundles in the case of zinc alone or glutathione. This may be due to the increased surface exposure of the elements. For the stem , as shown in table (4) and plate ( 3) It was found that there was a decrease in width of the cortex and concurrent with the decrease in number of rows of cortical cells and the number of vascular bundles with increased concentration of zinc alone or in combination with calcium nitrate or glutathione, noting that there is an improvement in the case of the addition of glutathione.This may be due to the fact that high concentrations of zinc may affect the rate of formation of auxins, which affects the rate of growth. On the other hand, Alpaslan et al. (1999) added that the addition of zinc to tomato plant with sodium chloride lead to increase the number of vascular bundles in the stem.
In leaf anatomy as shown in table (5) and plate (4), it was shown that, the thickness of the leaf in the zinc-treated plants alone was increased with increasing zinc concentration. This was coinciding with the increase in thickness of the midrib area and the expansion of the vascular bundle area.
With the addition of calcium nitrate, there was a decrease in these measurements with increasing zinc concentration. However, with the addition of glutathione, the thickness of leaf and a decrease in the mid rib area, while the thickness of the vascular bundle area did not show a change. Sidhar et al. (2007) suggests that, the, microscopic structural changes, such as a decrease in intercellular spaces, breakdown of vascular bundles, and shrinkage of palisade and epidermal cells, occurred in leaves, stems and roots of plants treated with high concentrations of Zn.
Shoots and roots of P. vulgaris seedlings seemed to show differential sensitivity to Zn stress. Reduction in shoot growth criteria seemed to result from a decrease in parenchyma cell size and diameters of metaxylem vessels in the leaf midrib. Scanning Electron Microscope (SEM) revealed the presence of compacted grana with reduced thylakoids in chloroplasts, which might have contributed to the recorded loss of chl-a, chl-b and carotenoids Kasim (2007)

Conclusion
Zinc added at the rates of, 200, 400, 600, 800, 1000 and 1200 mM ZnSO4. Maximum significant decrease in the growth affected the height of Phaseolus vulgaris plants significantly. At 1mM Zn, plant height was found to be highest (29.21 cm/plant) and then decreased with increasing Zn treatments. Fresh and dry matter production of Phaseolus vulgaris decreased with increasing Zn levels and found highest at 1 mM. Zn concentration in plants increased with increasing Zn treatment and was highest at 1200 mM in all treatments either in application of Zn alone or in combination with Ca +2 or with glutathione in both for root and shoot. There was a differential variation in anatomical structure of roots, stems and leaves owing to all treatments with zinc. Http://www.granthaalayah.com ©International Journal of Research -GRANTHAALAYAH [9] Table 2: Effect of different ZnSO4 concentrations in the culture medium either alone or in combination with (10mM) Ca (NO3)2 or glutathione on the zinc content in root ,in shoot and in the total uptake of 14-day-old Phaseolus vulgaris plants .Each value is the mean of 3 Sample calculated as m mole 100g-1 dry weight