AMELIORATING ROLE OF N.P.K. FERTILIZER ON THE TOXIC EFFECTS OF Ni ON (SORGHUM) ROOT ANTIOXIDANT ENZYMES

AMELIORATING ROLE OF N.P.K. FERTILIZER ON THE TOXIC EFFECTS OF Ni ON (SORGHUM) ROOT ANTIOXIDANT ENZYMES

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ABSTRACT

        This study investigated the activities of superoxide dismutase (SOD), catalase (CAT), glutothione peroxidase (GP) and the level of malondialdehyde (MDA) in the root of sorghum grown in soils contaminated with 30ppm nickel, 30ppm nickel +20ppm fertilizer and 30ppm nickel + 40ppm fertilizer.  Sixty sorghum seeds were germinated in these contaminated soils and  were harvested after 2 weeks, 3 weeks, and 4 weeks of planting.  Treatment of the plants with 30ppm nickel significantly increased (P < 0.05) the activities of SOD and the level of MDA in the roots compared with the controls.  Also, the treatment significantly decreased (P < 0.05) the activities of CAT and GP in the roots compared with controls.

        The study also revealed a significant decrease (P < 0.05) in the activities of SOD and the level of MDA in plants grown in 30ppm Ni + 20ppm NPK fertilizer and 30ppm Ni + 40ppm NPK fertilizer respectively compared with those grown in 30ppm Ni concentration.  These results show that 30ppm Nickel is toxic to sorghum roots for it increases significantly the production of reactive oxygen species but decreases significantly the excretion of reactive oxygen species.  This is due to significant increase in the activity of SOD but significant decrease in the activities of CAT and GP.  These results also showed that 30ppm Nickel damaged sorghum roots by significantly increasing lipid peroxidation and the levels of MDA.  In addition, the results revealed that 20ppm and 40ppm NPK fertilizer had ameliorating effect on the toxicity caused by 30ppm nickel.


CHAPTER ONE

1.0      INTRODUCTION AND LITERATURE REVIEW

1.1   INTRODUCTION

Trace metals are redistributed in environment by fossil fuel combustion.  This release can be expected to increase soil levels of trace elements such as Ni2+ resulting in a concomitant increase in the concentration of Ni2+ in plants and possibly in the food chain (Dominic et al, 1978). 

Nickel (Ni) is an essential micronutrient for plants since it is the active centre of the enzyme urease required for nitrogen metabolism in higher plants (Yan et al, 2008).  Nickel deficiencies lead to reduced urease activity in tissue cultures of sorghum, rice and tobacco and in excessive accumulation of urea and toxic damage to the leaves of leguminous plants such as sorghum (Peter and Andre, 1986).  However, excess Ni is known to be toxic and many studies have been conducted concerning Ni toxicity of various plant species. 

The most common symptoms of nickel toxicity in plants are inhibition of growth, photosynthesis, mineral nutrition, sugar transport and water relations (Seregin and Kozhevnikova, 2006).  Heavy metal affects plants in two ways.  First, it alters reaction rates and influences the kinetic properties of enzymes leading to changes in plant metabolism (Yan et al, 2008).  Second, excessive heavy metals lead to oxidant stress. 

During the period of metal treatment, plants develop different resistance mechanisms to avoid or tolerate metal stress, including the changes of lipid composition, enzyme activity, sugar or amino acid contents, and the level of soluble proteins and gene expressions.  These adaptations entail qualitative and/or quantitative advantage, and affect plant existence (Schutzendubel and Polle, 2002). 

It is known that excessive heavy metal exposure may increase the generation of reactive oxygen species (ROS) in plants, and oxidative stress would arise if the balance between ROS generation and removal were broken.  Oxidative stress is a part of general stress that arises when an organism experiences different external or internal factors changing its homeostasis.  In response, an organism either aims to maintain the previous status by activation of corresponding protective mechanisms or goes to a new stable state (Mittler, 2002). 

In several plants, Ni has been shown to induce changes in the activity of ROS – scavenging enzymes, including SOD catalase and glutathione peroxidase (Yan et al, 2008).

        The aim of this study is to investigate the effects of nickel on the activities of sorghum root antioxidant enzymes and also monitor the ameliorating effects of N.P.K. Fertilizer.

1.2      LITERATURE REVIEW

1.2.1        HEAVY METALS

        Heavy metal is any of a number of higher atomic weight elements, which has the properties of a metallic substance at room temperature.  There are several definition concerning which elements fall in this class designation.  One school of thought classifies metals having density greater than 5g/cm3 as heavy metals.  This classification includes most transition metals and higher atomic weight metals of Group III to V of the periodic Table. Examples of these heavy metals are zinc, cadmium, chromium, and Nickel.

1.2.2 CHARACTERISTICS OF NICKEL

        As with other metals, the biological significance, of Ni is related to its physicochemical properties some physical properties of Ni are shown in Table 1 below.  The preferred oxidation states of Nickel are O and +2 but in complexes +3 and +4 states can also occur (Sengar et al, 2008).  Nickel forms stable octahedral complexes with, for example, EDTA nitrolotriacetate, cysteine and citrate (Bagati and Shorthours, 1999).

Characteristics

Values

Atomic number

28

Atomic Weight

58.71

Boiling point

2913oC

Melting point

1455oC

General appearance

Soft silvery metal

Density of the metal

8.90

TABLE 1.0:  Physical properties of Nickel (Evenhort, 1971).

1.2.3               NICKEL IN THE ENVIRONMENT

Nickel is widely distributed throughout the physical and biological world.  In the soil, it is present in the form of its mineral ores; the important ones are Linnacite [(Fde. Co. Ni)3 S3] and spies cobalt [(Co. Fe. Ni)4 S2)].

The metal is extracted from its ore for various industrial, chemical and biological applications.  Natural weathering of igneous and metamorphic rocks also releases Ni, which is largely retained in the weathered profile in association with clay minerals and as hydrous ions or as a complex with manganese oxide.  Free Ni concentration in the soil is controlled primarily by precipitation reactions with the hydrous oxides of Mn and Fe metals.  Nickel also occurs in water bodies and in atmosphere, usually in trace amounts.  The relatively higher concentration of Ni in sediments indicates that the metal gets deposited by the physicochemical reactions in water and in riverbed (Sengar et al, 2008).  This is apparently favoured by alkalinity and high oxides of other co-precipitating metals (Israili, 1992).

1.2.4        BIOLOGICAL ROLES OF NICKEL

i.      SEED GERMINATION AND SEEDLING GROWTH

It has been observed that 2ppm solution of Ni(NO3)2 and NiSO4 accelerated the germination of wheat grains.  When used as a pre-sowing treatment, NiSo4 solution in the concentration range of 2.68 to 26.3ppm had a marked stimulating effect on the germination of pea (pisum sativum), bean (Phaseolus vulgaris), wheat and castor seeds (Underwood, 1971). The germination of rice seed and the activities of some oxidative enzymes in the seedlings were stimulated by 3ppm Ni (Bushnell, 1966). According to Welch (1981), the stimulation of germination by Ni (Pelosi et al, 1976) may be based on the function of Ni as the metal component of urease.  After studying the effect of 0.1mM concentrations of Ni on seedling growth and activities of certain hydrolytic enzymes of seeds of phaseolus aureus, Veer (1988) suggested that Ni inhibits seedling growth by the suppression of the activities of hydrolytic enzymes (Walker et al, 1985).

ii.     CO-FACTOR:

Studies have shown that nickel is an essential cofactor required by some enzymes particularly urease (Eskew et al, 1983).  In their work, soybean plants deprived of nickel accumulated toxic concentrations of urea (2.5%) in necrotic lesions on their leaflet tips.  This occurred regardless of whether the plants were supplied with inorganic nitrogen or were dependent on nitrogen fixation.  Nickel deprivation resulted in delayed nodulation and in reduction of early growth. Addition of nickel (1ug/L) to the nutrient media prevented urea accumulation, necrosis and growth reduction.

1.2.5        ABSORPTION OF NICKEL BY PLANTS

        A number of reports (Aschmann and Zasoski, 1987; Crooke et al, 1954) indicate that Ni is easily absorbed by the plants when supplied in the ionic form (Ni21) and is not as strongly absorbed when chelated. Turina (1968) reported that in some monocats like rye (Secale Cereale), wheat (Triticum Vulgare) and maize (zea mays), the absorption of Ni by roots was through the root caps.  Ni uptake appears to be an active process, as it is influenced by temperature and anaerobic condition and by respiratory inhibitors such as dinitrophenol (Aschmann and Zasoski, 1987).  Dominic et al (1978) reported that the absorption of Ni2_ by intact soybean plant and its transfer from root to shoot were inhibited by the presence of cu2=, zn2+, Fe2+ and Co2+. Competition kinetic studies showed Cu2+ and Zinc to inhibit Ni2+ absorption competitively, suggesting that Ni, Cu2+ and Zn2+ are absorbed using the same carrier site.  Calculated Km and Ki constants for Ni2+ in the presence and absence of Cu2+ were 6.1 and 9.2uM, respectively, whereas Km and Ki constants were calculated to be 6.7 and 24.4uM, respectively, for Ni2+ in the presence and absence of Zn2+.  A number of reports have also shown that plants uptake of Ni2+ depends on its ionic form and Ni concentration in the medium (Dixon et al, 1980; Miller, 1961).  The absorption of Ni is also increased by increasing the phosphate content of the soil (Halstead et al, 1969; Polacco, 1976).  Fertilizers also decreases the total absorption of Ni.  Nickel has also been shown to be easily taken up from acidic soil solutions by plant roots and is transported in free and chelated forms to the transpiring leaves via the xylem  (Peter and Andre, 1986).  Ni ions are reportedly less available in the roots of plants growing on alkaline soils and these plants might therefore be subject to suboptimal rates of supply from the soil (Peter and Andre, 1986).

1.2.6  ACCUMULATION OF NICKEL IN PLANTS

During vegetative growth, most of the Ni is translocated and accumulated in leaves. However, during senescence of leaves, most of it is transported to seeds, as reported for soybeans (Cataldo et al, 1978).  Studies on the chemical forms on Ni in plants tissues have shown that the metal is present in the form of a cationic complex (Krog Niel et al, 1991; Mishra and Kar, 1974).  A large number of plants have been identified as Ni2+ phytoremediator including Indian mastered fragnant geranium sunflower, Thlaspi sp. Alyssum (Cunningham et al, 1995) Berkheya coddii (Kramer et al, 1996) sebertia acuminatav (Ensley et al, 1997).

1.2.7  NICKEL AND PHOTOSYNTHESIS

The metal is known to inhibit photosynthesis and overall gas exchange in some plants such as maize and sunflower (Lo and Chen, 1994; Mishra et al, 1973).

Sheoran and Singh (1993) have suggested that the metal inhibit photosystem (Ps) II more effectively possibly at the oxidizing site.  Long term exposure of Ni to plants has been shown to result in reduced leaf growth, decreased photosynthetic pigments, changed chloroplast structure and decreased enzyme activities for CO2 assimilation (Dan et al, 2000).

1.2.8  EFFECTS OF NICKEL ON PLANT RESPIRATION

The rate or respiration in the healthy tissues of wheat leaves increases on treatment with Ni salts (Aschmann and Zasoski, 1987); Ensley et al, 1997).  Miller et al (1970) demonstrated that NicL2 at lower concentration increased the respiratory rate of maize mitochondria but at high concentrations, the respiratory reaction was blocked.  The concentration of Ni producing maximal respiratory response is 4.7ppm NiSO4. The report of Miller et al (1970) further indicated that at 5.87ppm Ni SO4 increased the NADH – oxidation in the absence of phosphate by about 5%.

1.2.9        METABOLIC EFFECTS OF NICKEL

        One of the most obvious effects of Ni supply has been on the protein metabolism.  Nickel increased the total protein content and total nitrogen content of maize and oat plants (Mishra and Kar, 1974; Welch, 1981).  Spraying of infected plants with NiSO4 solutions at the stage of 5 – 6 leaves increased the free amino acid content of the leaves (Borrks and Marfil, 1981).  Lo and Chen (1945) have reported that NiSO4 in combination with complete fertilizers increases the ascorbic acid content of phaseolus Lactuca and Tomatoes (Alagna et al, 1984).

1.2.9  EFFECTS OF NICKEL ON ENZYME ACTIVITY

Several investigators have measured the activities of enzymes in response to Ni.  Ni plays a significant role in enzyme catalysed metabolic processes often functioning as a cofactor, as is evident from Table 1.1 below.  Nickel is not required for the synthesis of the enzyme protein but as metal component, it is essential for the structure and functioning of enzyme (Klucas et al, 1983; Roach and Barcloy, 1946).

1.2.11  MECHANISM OF NICKEL TOXICITY

        A number of mechanisms have been proposed to account for the toxicity of nickel.  Although, some of the mechanisms presented in this review have been demonstrated in animal studies, they could also account for the toxicity of this heavy metal in plants:

i.            PRODUCTION OF REACTIVE OXYGEN SPECIES

Nickel stress can lead to the production of such reactive oxygen species (ROS) as hydroxyl radical (OH) and superoxide anion (02) (Schutzendubel and Polle, 2002).

ii.          INHIBITION OF ROOT BRANCHING

Seregin and Kozheunikova (2006) have reported that a high nickel content in the endoderm and pericycle cells blocks cell division in the pericycle and results in the inhibition of root branching.

iii.       ANTAGONISM OF Mg2+

Pane et al, (2003) reported that the clearest effect of nickel exposure on Daphnia magna was an Mg2+ homeostasis.  They reported that the concentration of whole body Mg2+ was significantly decreased by 18% following acute and chronic exposure.

iv.         REPLACEMENT OF DIVALENT CATIONS AT ACTIVE SITE OF ENZYMES

Nickel can replace Zn2+, Co2+ or any other heavy metal present at the active site metallo-enzymes and disrupt their functioning.

v.           PRODUCTION OF STRESS ETHYLENE

It is well documented that plants respond to a variety of different environmental stresses by synthesizing “stress” ethylene (Abeles et al, 1992).  A significant portion of the damage to plants from environmental stress may occur as a direct result of the response of the plant to the increased level of stress ethylene (Van Loon, 1984).  Van Loon (1984) noted that in the presence of fungal pathogens, not only does exogenous ethylene increased the severity of a fungal infection but also inhibitors of ethylene synthesis can significantly decrease the severity of infection.

This research finding, in addition to the finding that the enzyme ACC deaminase, when present in plant growth promoting bacteria, can act to modulate the level of ethylene in a plant prompted Burd et al (1998) to find out if such bacteria might lower the stress placed on plants by the presence of heavy metals and therefore ameliorate some of the apparent toxicity of heavy metals to plants.

1.2.12            STRATEGIES OF PLANT TOLERANCE TO Ni TOXICITY

Plants have several strategies they adopt to mitigate the effects of high concentrations of heavy metals.  Some of the strategies they employ in the face of nickel stress are:

1.          Increase in the activity of peroxisomal H202 scavenging enzymes (Gonnelli et al, 2001).

2.           INCREASE IN THE INTRACELLULAR CONCENTRATION OF GLUTATHIONE

Freeman et al, (2004) reported that concentrations of glutathione, cysteine and O-acetyl-L-serine (OAS) in shoot tissue, are strongly correlated with the ability to hyperaccumulate nickel in various Thlaspi hyperaccumulators collected from serpentine soils, including Thlaspi goesingense, T. oxyceras, and T. rosulare, and nonaccumulator relatives, including T. perfoliatum, T. arvense, and Arabidopsis thaliana.

A nearly ubiquitous antioxidant, glutathione plays a critical role in minimizing oxidative stress, or damage caused by highly reactive compounds.  Plants require metals like nickel in minute quantities for certain metabolic processes, but at high levels metals can damage membranes, DNA and other cell components.  Most plants try to keep the levels of metals in their cells at a minimum but plants called metal hyperaccumulators have the unique ability to build up unusually high levels of metals in their tissues without any ill effect.  Previous research indicates that hyperaccumulators store metals in a specialized cell compartment called the vacuole.  Sequestered in the vacuole, nickel and other metals can’t damage other parts of the cell.  But nickel still must travel within the cell in order to enter the vacuole in the first place.

To get to the vacuole, the nickel has to traverse the interior of the cell, where most of the plant’s sensitive biochemical processes reside.  So Freeman et al (2004) set out to find out if there’s something in the cell’s interior that protects it from oxidative damage as the metal crosses the cell.

In this study, Freeman and his colleagues sampled a number of closely related plants that grow on soils naturally enriched in nickel.  These plants ranged from those that didn’t accumulate any nickel to the hyperaccumulators that built up almost 3% nickel – by weight. They found that the concentration of glutathione was well correlated with a plant’s ability to accumulate nickel.  The next step was to establish that glutathione played a functional role in nickel tolerance.  He and his colleagues isolated a gene called SAT, and inserted it into a model lab plant called Arabidopsis thaliana, which does not normally tolerate nickel.  The gene SAT produces an enzyme called serine acetyltransferase, which plays a role in producing glutathione in hyperaccumulating plants.

When Freeman and his colleagues  grew both normal Arabidopsis and those containing the SAT gene on a nickel – containing medium, the normal plants failed to grew and showed signs of severe membrane damage, an indicator of oxidative stress.  The plants with the inserted gene thrived, showing no signs of membrane damage.

Going one step further, Freeman and his colleagues conducted another experiment in which they exposed the Arabidopsis containing the SAT gene to a compound that inhibits their ability to make glutathione.  When grown on nickel, these plants also suff


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