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

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.

TABLE OF CONTENTS

Title Page i

Certification ii

Dedication iii

Acknowledgement iv

Table of Contents vi

Abstract ix

CHAPTER ONE

1.0 Introduction and Literature Review 1

1.1 Introduction 1

1.2 Literature Review 3

1.2.1 Definition of heavy metals 3

1.2.2 Characteristics of Nickel 4

1.2.3 Nickel in the environment 5

1.2.4 Biological roles of nickel 6

1.2.5 Absorption of nickel by plant 7

1.2.6 Accumulation of Nickel in plants 9

1.2.7 Nickel and photosynthesis 10

1.2.8 Effects of nickel on plant respiration 10

1.2.9 Metabolic effects of nickel 11

1.2.10 Effects of nickel on enzyme activity 11

1.2.11 Mechanism of nickel toxicity 12

1.2.12 Strategies of plant tolerance to nickel toxicity 14

1.2.13 Management of nickel toxicity 18

1.2.13.1 Land management procedure 18

1.2.13.2 Phytoremediation 19

1.2.14 The use of micro-organisms to mitigate nickel toxicity 22

1.3 Scientific classification of Sorghum 23

1.4 Chemical Composition and Nutritive Value of

Sorghum 25

1.5 Classification of sorghum 28

1.6 Uses of Sorghum 29

1.7 Germination / Growth Stages of Sorghum 32

1.7.1  Growth Stages 32

1.72 Nutrient Uptake 36

1.8 Diseases of Grain Sorghum 37

1.9 Activities that induce Germination 38

1.10 Metabolism of Germinating Seeds 40

1.11 NPK (15-15-15) Fertilizer 41

1.11.1Catalase 44

1.12 History of Catalase 45

1.12.1  Activities of Catalase 46

1.12.2 Molecular mechanism of catalase action 47

1.13 Superoxide Dismatase 48

1.13.1Types of Superoxide Dismutase 49

1.13.2 Physiological Importance of Superoxide Dismutase 51

1.13.3 Use of Superoxide Dismutase in Cosmetic 52

1.14 Peroxidase 52

1.14.1 Isozymes of Glutathione Peroxidase 53

1.15 Oxidative stress and reactive oxygen species 53

1.16 Objective of the Study 56

CHAPTER TWO

2.0 Materials and Method 55

2.1 Materials 55

2.1.1 Contaminant 55

2.1.2 Fertilizer 55

2.1.3 Quantity of soil used 55

2.1.4 Source of Soil 55

2.1.5 Source of Soybean seed used 56

2.1.6 Instruments/Apparatus used 56

2.1.7 Reagents used for the study 57

2.2 Methods 59

2.2.1 Preparation of Soil 59

2.2.2 Contamination of Soil 59

2.2.3 Viability test of Seeds 59

2.2.4 Experimental design 59

2.2.5 Biochemical analysis 62

2.2.5.1 Estimation of total protein 61

2.2.5.2 Estimation of malondialdehyde level 64

2.2.5.3 Estimation of Superoxide Dismutase activity 66

2.2.5.4 Estimation of Catalase activity 68

2.2.5.5 Estimation of peroxidase activity 70

2.2.6   Statistical Analysis 72

CHAPTER THREE

3.0 Results 73

3.1 Soil Analysis 78

CHAPTER FOUR

4.0 Discussion and Conclusion 79

Bibliography 83

Appendix One: Reagents Preparation 97

Appendix Two:  Statistics 101

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 suffered high levels of oxidative damage just like their normal counterparts.  This conforms that it really is glutathione that’s responsible for nickel tolerance.

iii. Complexing of the nickel to organic molecules.

1.2.13 MANAGEMENT OF NICKEL TOXICITY

There are a number of strategies that can be adopted to reduce the concentrations of nickel in the soil that is available to plants for absorption.

1.2.13.1 LAND MANAGEMENT PROCEKDURE

This involves the application of chemicals to nickel – contaminated soils with the aim of reducing the ability of economically important plants to absorb the contaminant from the soil.  Robinson et al (1999) noted that treatment of serpentine soils with certain chelating agents caused a significant reduction in plant uptake of nickel, despite increasing the solubility (plant availability) of this element – in the soil.  They used pet trials to investigate the effects of MgC03, EDTA and citrate, CaC03, sulphur and acid mine tailings on nickel and cobalt uptake by the South African nickel hyperaccumulator Berkheya coddii.  Robinson et al (1999) grew plants in nickel rich ultramafic (‘Serpentine’) soil diluted with pumice.  Both MgC03 and CaC03 caused significant decreases in the uptake of both metals, as well as decreasing their solubility in the soil.  After the addition of MgC03, there was a significant increase in soil pH, So the reduction in plant-metal uptake could not be solely attributed to the action of magnesium alone.  Since CaC03 had no significant effect on soil pH, this indicated that calcium inhibits the uptake of both cobalt and nickel.

1.2.13.2 PHYTOREMEDIATION

This is the use of green plants to remove or render harmless environmental contaminants like nickel.  It is considered to be an attractive alternative to the approaches that are currently in use for dealing with heavy metal contaminants (Cunningham et al, 1995).  Phytoremediation of metals might take several forms:

i. Phytoextraction:  This refers to processes in which plants are used to concentrate metals in the roots and shoots of the plant.

ii. Rhizofiltration:  This is the use of plant roots to remove metals from effluents.

iii. Phytostabilization:  This is the use of plants to reduce the mobility of heavy metals (and thereby reduce the spread of these metals in the environment)t).

Suresh and Ravishankar (2004) have identified phytoremediation as an environmentally friendly approach for remediation of contaminated soil and water using plants.  According to them, phytoremediation has two components:

i. One by the root colonizing microbes and the other,

ii. By plants themselves, which degrade the toxic compound to further non-toxic metabolites.

Various compounds, viz; organic compounds l, xenobiotic, pesticides and heavy metals, are among the contaminants that can be effectively remediated by plants.  Plant cell cultures, hairy roots and algae have been studied for their ability to degrade a number of contaminants.  They exhibit various enzymatic activities for degradation of xenobiotics, viz. dehydrogenation, dinitrification leading to breakdown of complex compounds to simple and non-toxic products.

Plants and algae also have the ability to hyperaccumulate various heavy metals by the action of phytochelatins and metallothioneins forming complexes with heavy metals and translocate them into vacuoles. Molecular cloning and expression of heavy metal-accumulator gene and xenobiotic degrading enzyme – coding genes resulted in enhanced remediation rates, which will be helpful in making the process for large – scale application to remediate vast-areas of contaminated soils.

A few companies worldwide are also working on this aspect of bioremediation, mainly by transgenic plants to replace expensive physical or chemical remediation techniques:

i. Selection and testing of multiple hyperaccumulator plants.

ii. Protein engineering of phytochelatins and membrane transporter genes and their expression.  

These procedures/techniques could make the process of phytoremediation a successful one for bioremediation of environmental contamination (Suresh and Ravishankar, 2004).

1.2.14 THE USE OF MICRO-ORGANISMS TO MITIGATE NICKEL TOXICITY

Burd et al (1998) have reported that the bacterium Kluyvera ascorbata SUD165 is highly effective at protecting plants from growth inhibition caused by the presence of high concentrations of nickel.  However, on a dry-weight basis, the plant grown in the presence and absence of the bacterium took up approximately the same amount of nickel, so that it is unlikely that the bacterium limits the uptake of nickel by the plant.

The most likely explanation of the data obtained by Burd et al (1998) is that the bacterium protects the plant against the inhibitory effects of nickel-induced stress ethylene production. In this regard:

i. Heavy metals can induce ethylene production by plants (Weckx et al, 1993).

ii. An excess of ethylene can inhibit plant development (Jackson, 1991), and

iii. The direct promotion of plant root growth by a number of different soil bacteria is based on the ability of bacteria ACC deaminase to hydrolyse and decrease the amount of ACC – the precursor of ethylene – in plants and, as a result, to decrease ethylene biosynthesis by plants (Glick et al, 1998).

1.3 SCIENTIFIC CLASSIFICATION

Kingdom Plantae (Plants)

Subkingdom Trancheobionta (Vascular plant)

Super division Spermatophyta (Seed plant)

Division Magnoliophyta (Flowering plant)

Class Liliopeida (Monocotyledon)

Subclass Commelimidae

Order Cyperales

Family Poacease (Grass family)

Subfamily Panicoideae

Germs Sorghum

Specie Sorghum  Bicolor(L)

Sorghum is one of the most important cereal crops grown in Nigeria, with species of about 30.  Sorghum bicolour (grain sorghum) is the primary cultivated species.  It has a chromosome number of 10 (2n = 20) and a C4 photosynthetic pathway (Ammuganathan and Earle, 1991).  Grain sorghum is an annual grass similar in appearance to maize (Corn), although, it has more tillers (stems) and more finely branched roots. Wild sorghum is a tall plant of 5 – 7 feet.  Though, newer varieties which are about 2 – 4 feet tall are now produced having 2 – 3 dwarf genes thus making harvest easier.  On the panicle, the spiklets are in pairs and bear white, yellow and brown grains.  The browner seeds are higher in tannins.  When the main panicle is damaged, branches can produce grain (Carter et al, 1989, Crop Plant Resources, 2000). The nutritional status of sorghum indicates that it is rich in carbohydrate, dietary fibre, protein, fat and fatty acid, Vitamins (Thiamin, Riboflavin, Niacin, etc), Minerals (Calcium, magnesium, phosphorus, potassium, sodium, etc) and amino acids (lysine, threonine, valine, methionine and cysteine, isoleucine, leucine, phenylalanine and tyrosine etc.).

1.4 CHEMICAL COMPOSITION AND NUTRITIVE VALUE OF SORGHUM

The sorghum grain is low in protein and ash and rich in fibre components.  The germ fraction in sorghum is rich in ash,  protein and oil but very poor in starch.  Over 68% of the total mineral matter and 75% of the oil of the whole kernel is also rich in B-complex vitamins.  Endosperm, the largest part of the kernel, is relatively poor in mineral matter, ash and oil content. It is however a major contributor to the kernel’s protein (80%), starch (94%) and B-complex vitamins (50-75%).

Like other cereals, sorghum is predominantly starchy.  The protein content is nearly equal among other grains and is comparable to that of wheat and maize.

Table 1.4.1 Nutrient content of whole kernel and its fractions

Kernel fraction % of kernel wt Protein (%) Ash (%) Oil (%) Starch (%) Niacin (mg/ 100g) Riboflavin (mg/ 100g) Pyridoxin

Mg/100g)

Sorghum Whole Kernel 100 12.3 1.67 3.6 13.8 4.5 0.13 0.47

Endosperm 82.3 12.3 0.37 0.6 82.5 4.4 0.09 0.40

(80)(20)(13)(94)(46)(50)(76)

Germ 9.8 18.9 10.4 28.1 13.4 8.1 0.39 0.72

(15)(69)(76)(20)(17)(28)(16)

Bran 7.9 6.7 2.0 4.9 34.6 4.4 0.40 0.44

(4.3)(11)(11)(4)(7)(22)(8)

A= Values in parenthesis represent % of whole, and value 

B  =  N x 6.25

(Hubbard, Hall and Earle.Sorghum, 1950).

Other nutrient composition of Sorghum include:

Fat (g)  =  3.1 amylose (%)  =  24.0

Energy (Kcal) = 329 gelatinization temp (%) = initial = 

68.5, final = 75.0

Ca (mg) = 25 water binding capacity (%)  =  105.

Fe (mg) = 5.4

The germ and aleurone layers are the main contributors to the lipid fraction.  The germ itself provides about 80% of the total fat.  The mineral matter is more concentrated in the germ and seed-coat.  The average starch content of sorghum is 69.5%, about 70 – 80% of the sorghum starch is amylopectin and the remaining 20 – 30% is amylose.  All sorghum contains phenol and most contain flavonoids, sorghum containing tannin (tannin or bran sorghum contains tannins even though the pericarp colour may be white, yellow or red as grain appearance does not necessary relates to tannin presence.  Brown sorghum contained the highest amount of free phenolic acids, and  resistant to fungal attack contained both a greater variety and larger amount of phenolic acids in free form.

Table 1.4.2 Free and Bound Phenolic Acid Composition (ug/g) of Sorghum

Phenolic acid White sorghum Red Sorghum Brown sorghum

  FREE BOUND FREE BOUND FREE BOUND

Gallic - 19.7 - 46.0 - 26.1

Protocatechol 7.4 133.9 13.0 83.0 8.0 15.8

P-Hydroxybenzoic 4.0 11.4 6.7 16.0 9.3 24.2

Vanillic 8.3 - 7.7 19.2 23.3 27.4

Caffeic 3.4 22.2 4.1 48.0 8.7 26.8

P-Coumaric 95.7 138.5 13.5 72.5 6.4 79.9

Fenilic 45.4 27.2 8.9 95.7 26.0 91.9

Cinnamic 9.4 - 10.7 - - 19.7

(Hahn et al, 1983)

Tannins protect against insect, birds, fungi and weathering.

1.5 CLASSIFICATION OF SORGHUM

Sorghum is a genus with many species and subspecies and there are several types of sorghum which can be classified into four groups.

GRAIN SORGHUM:  These are grown for their grain round, starchy seeds used as human food or cattle feed.

GRASS SORGHUM:  Grown for their green feed (forage) and making silage or hay (dried fodder).

SWEET SORGHUM:  Grown for their juicy stems and are grown for making sorghum syrup and also for animal feed.

BROOM CORNS:  Grown for the branches of the seed cluster which are used to make brooms.

Grain sorghum requires less water than corn and thus grown as a replacement for corn and produces better yield than corn in hotter and drier areas such as Southern US, Africa, Central America and South Asia i.e. to say, it is cultivated in warmer climates worldwide.  Most species are drought tolerant and heat tolerant.  Like maize, it can be grown under irrigation. It’s propagated by seed and responds well to inorganic NPK fertilizer.  The fertilizers are incorporated into soil at or prior to planting.

1.6 USES OF SORGHUM

Sorghum like many grains has diversity of uses thus has it is placed as the fifth most important cereal crop grown in the world with Nigeria producing 14% of the world sorghum production in 2005, making it the second largest sorghum producer in the world producing 1,000,000 – 9,999,999 metric tones of sorghum.  Some of its uses include (Carter et al, 1989, Crop Plant Resources, 2000).

a. IN HUMAN FOOD

Sorghum is used as food for human nutrition all over the world – grain sorghum is used for flours, porridge and side dishes, malted and distilled (alcoholic) beverages and speciality food such as popped grain (pop corn).  Sorghum species are important food crops in Africa, in China, sorghum is fermented and distilled to produce “MAOTI” which is regarded as the country’s most famous liquor.

b. IN ANIMAL FEED

Sorghum is also considered a significant crop for animals feeds.  Grain sorghum is also used for silage, but sweet sorghum have higher silage yield.  Some species of sorghum can contain level of hydrogen cyanide (HCN), prussic acid, hordenine and nitrates lethal to grazing animals in the early stage of plant growth.

c. OTHER USES

The reclaimed stalk of sorghum plants are used to make a decorative mill work material marketed as “Kirei board”, its fibre are also used for fences, biodegradable packaging materials and solvents.  Dried stalks are used as cooking fuel and dye can be extracted from the plant to colour leather.  A more recent use of sorghum is for ethanol, sorghum is also used in malt production used as a replacement for barley malt in beer manufacture.  In India and other places, sweet sorghum is also used in malt production used as a replacement for barley malt in beer manufacture.  In India and other places, sweet sorghum stalks are used for producing biofuel by squeezing the juice and then fermenting into ethanol.  The United States is currently running a trial to produce the best varieties for ethanol production from sorghum leaves and stalks.

1.7 GERMINATION/GROWTH STAGES OF SORGHUM

1.7.1 GROWTH STAGES

Growth stage Approximate days Identifying 

after emergence characteristics

00Emergence celeoptile

at soil surface

110

4 40 Collar of 3rd leaf visible

220Collar of 5th leaf visible

330Growing point 

differentiation Final leaf visible in whorl

5 50 Boot head extended 

Into Flag leaf sheath

6 60 Half-bloom

7 70 Soft dough

8 85 Hard dough

9 100 Physiological maturity

maximum dry matter accumulation

(Vanderlip, R.L. 1993).

STAGE O: Emergence, when the sorghum seed is sown in moist soil, the seed swells due to moisture absorption.  The seed coat breaks a small shoot (coleophile) and a primary root (radicle) emerge, germination occurs 3 – 10 days after planting.  During this period, growth depends on the seed (endosperm) for nutrients and food reserves.  The key to a reliable sorghum germination depends on an adequate soil temperature, moisture condition, depth of planting and vigor of seed.  Cool, wet conditions during this time may favour disease organism which can cause damage.  Optimum grain sorghum seedling depth is 1.25 – 1.5” (inches) deep and the minimum soil temperature required for sorghum germination is 16 – 17oC (65oF) with quick germination process and the longer it takes a seedling to emerge, the greater the risk being attacked by soil borne diseases.

STAGE 1

Three leaf stage – leaves are counted when the collar (i.e. the place where the leaf blade and leaf sheath attach) of the leaf can be seen without tearing the plant apart.  Secondary root begins to grow.

STAGE 2

Approximately 3 weeks after emergence, a sorghum plant has five leaves fully expanded, its root system developing rapidly.  The plant enters its “grand period of growth” in which dry matter accumulate at nearly a constant rate until maturity, if growing conditions are satisfactory.  The sorghum root system consists of three types of roots which are primary root (which develops from the radicle) secondary or adventitious roots (which develop from the first node from the mesocotyl) and Brace or buttress roots (which develop from the root primordial of the basal nodes above the ground level).

STAGE 3  

Growing point differentiation – About 30 days after sorghum emerges, its growing point changes from vegetative (leaf producing) to reproductive (head producing).  Nutrient uptake is rapid and stalk growth increases rapidly.

STAGE 4  

Flag leaf (final leaf) is visible in whorl, head is developing growth and nutrient uptake is rapid, almost all leaves expanding to full size.

STAGE 5

All leaves are fully expanded producing maximum leaf area and light interception.  Peduncle elongation is beginning which will result in the exertion of the head from the flag – leaf sheath.

STAGE 6

Half blow-following the book stage, the peduncle grows rapidly extending the head through the flag sheath. Half-bloom is usually defined as when one half of the plants in a field or area are in some stage of boom. However, because an individual sorghum head flowers from the tip downwards, half-bloom on an individual plant is when the flowering has progressed half way down the head.  At half bloom, approximately one-half of the total dry weight of the plant has been produced with nutrient uptake reaching nearly 70, 60 and 80% of total N, P and K respectively.

STAGE 7 

Soft dough – the grain fills rapidly and as a result the colon or stalk loses weight, lower leaves are being lost with 8 – 12 functional leaves remaining during this stage.

STAGE 8  

Hard dough – about ¾ of the grain dry weight has accumulated.  Nutrient uptake is essentially complete.

STAGE 9  

Physiological maturity – maximum total dry weight of the plant has occurred.  Physiological maturity can be determined by the dark spot on the opposite side of the kernel from the embryo (Vanderlip, 1993).

1.7.2 NUTRIENT UPTAKE

Nutrient uptake precedes dry matter accumulation because nutrients are required for growth and dry matter accumulation   Potassium is taken up most rapidly followed by nitrogen then phosphorus.  Large quantities of nitrogen and phosphorus and some potassium are translocated from other plants to the grain as it develops. Most of the potassium is in the vegetative part of the plant (Vanderlip, 1993).

1.8 DISEASES OF GRAIN SORGHUM

Seed may be attacked by one or more seed-borne or soil-borne pathogens prior to germination or emergence.  This usually occurs when conditions are not optimum for plant development such as poorly-drained, cold, wet soil, or even in very dry, crusted soils, as these conditions are generally favourable both for pathogen activity and for disease development.

ROOT ROTS

This is caused by fungi such as periconia circinata and species of pythium, rhizoctonia and fusarium. It can be controlled by management practices which stimulate plant vigour and development.

STALK ROTS

These include charcoal rot caused by the soil-borne fungus Macrophomina phaseolina, it causes the interior of the stalk to be shredded.  It can be controlled by giving disease resistant varieties and applying proper management practices such as balanced soil fertility (avoiding high levels of N and low level of potassium) avoid excess planting populations, irrigation, irrigate during dry periods after heading etc.

Other fungi diseases include Northern leaf blight gray leaf spot, southern leaf blight, Head diseases caused by Fugarium Curvulain etc.  Striga Hermonthica is a major biotic constraint to sorghum production in Nigeria, recommendations for Striga management often includes the use of cultural and agronomic practices, herbicides and host plant resistance when available.  Head smut caused by Sphaccolotheea Reliana in which the entire sorghum head is converted to sorus and then decay may be controlled and prevented by the use of resistance varieties and treatment with fungicides (Marley, et al, 2004).

1.9 ACTIVITIES THAT INDUCE GERMINATION

Germination occurs when some conditions that stimulate growth is resting on a seed.  Some of these conditions include exposure to moisture, adequate aeration and appropriate temperature.

The first stage in the process of germination is the rapid uptake of water at a high rate which decreases with time.  In the dry seed, the respiration rate is extremely low but the absorption of water immediately promotes and enhances gaseous exchange.  The promotion of metabolic activities in a dry grain is probably the result of a change in few conditions (Steward, 1999):

i. Changes in the activities of enzymes that are present in the dry seed.

ii. Changes in the structure of the cytoplasm that sustains metabolic activity.

iii. Change in metabolic patterns as a result of a change in quantitative relations between enzymes in the growing system.

iv. Change in synthesis of enzyme before and after growth has begun.

In addition, germination of seeds is regarded as a step that stimulate a quiescent seed with low metabolic activity into an actively metabolising tissue leading to the formation of seedlings from the embryo (Chandria and Dugnestes, 2000).

1.10 METABOLISM OF GERMINATING SEEDS

The hydration of proteins in germinating seeds begins with a large number of enzyme catalysed reactions.  Some of these enzymes that are present in dry seeds and are activated and synthesized de novo (Bewley and Black, 1978). Dry seeds posses primarily B-amylase. The x-amylase activities in most dry seeds rises sharply (Dave, 1999).  Other enzymes attacking starch are also released as germination progresses.  Starch and dextrin decreases with germination in the endosperm as well as in the whole grain.  Fat is decreased in the first few days and increased later.  The resynthesis of fat, appearance of sugar is the result of transformation of the disappearance storage carbohydrate (Opoku et al, 2000).

The proteolytic activities of protease which degrades proteins into constituent amino acids in germinating seeds have been reported (Jacobson and Verner, 2001).  The levels of these enzymes are under the regulation of plant growth hormones (Bewley and Black, 1978). Lipolytic activities have been demonstrated in germinating seeds (Mayer and Marback, 1999).  This is rapid conversion of fat to carbohydrate during germination.  This conversion is via the glyoxylate pathway, which is characterized by two enzymes, malate synthase and isolitrate lyase.

Germination and seedling emergence have a high demand for energy via respiration.  The energy released by digestion and oxidation phosphorylation is used to produce energy rich nucleotides such as ATP in the mitochondria. The ATP produced is then used to drive biological activities as shown below:

ATP  ATP

Carbohydrate         Degradation Biosynthesis 

Orproducts   of fat

Protein

ADP + PiADP + Pi

1.11 N.P.K. 15-15-15 FERTILIZER

N.P.K. fertilizer is a compound fertilizer.  Nutrients are recycled and made available to organisms on a continuous basis when these communities are replaced by cultivated crops.  The situation changes drastically, firstly, soil is much exposed to erosion and the loss of nutrients which may be removed with crops themselves or with animals with which the crops are fed.  The most important minerals nutrient that needs to be added to the soil are nitrogen, phosphorus and potassium.  All of these elements are needed in large quantities and are most likely to be deficient in soil.  But because they are often added in large quantities, they can also be significant sources of pollution in certain situations.  Commercial fertilizers are normally assigned a grade that reflects the percentages of nitrogen (N), phosphorus (P) and potassium (K), they contain by dry weight.  Thus, 15-15-15 means 15% of each of these elements (N, P and K) in the fertilizers (Raven, 2000).  The optimal proportions are best determined by testing the fertilizer of the soil and knowing the requirement of the particular crop or plant to be grown.

Nitrogen is usually supplied to crops in very large amounts since it seems to have a major effect on crop yield.  Nutrients other than these three may also be scarce in specific soils but such situations are rare and must be dealt with individually (Johnson et al, 1999).

In N.P.K. 15-15-15 fertilizer, the basic nutrients are contained in a form which is easily acceptable by plants.  The nitrogen here is approximately 40% in the form of the nitrate and of 60% in the ammoniacal form, more than 40% of phosphorus is in the water soluble form of the chloride.  The granulation of the fertilizer ensures a quick and exact dosing.

1. Total nitrogen content in % 15

1.1 Nitrate (N) content in % 6.7

1.2 Ammonium (N) content in % 8.3

2 Content of phosphates soluble in neutral ammonium citrate as p205 15

2.1 Content of phosphates soluble in water as P2O5 in % 8.5

3.0 Total potassium content as K20 in % 15

4.0o Total nutrient content as N + P205 + K20 in % 45

Table 1.3:  Technical parameters

N.P.K. 15-15-15 fertilizers is a compound fertilizer that is suitable for soils with medium and higher demand for nitrogen and phosphorus.  It is the most widely spread European type of compound fertilizer particularly in Germany and Australia.  N.P.K. 15-15-15 may be dangerous to health if swallowed or in contact with the mucous membranes, eyes and repeated contacts with the skin.  The fertilizer powder is irritable and may cause an over sensitiveness or eczemas (Brown and Martha, 2000).

1.11.1 CATALASE

Catalase (hydrogen peroxide oxido-reductase) is a common enzyme found in nearly all living organisms which are exposed to oxygen, where it functions to catalyse the decomposition of hydrogen peroxide to water and oxygen (Chelikain et al, 2004). Catalase has one of the highest turnover number of all enzymes; one molecule of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen per second (Goodsell, 2004). Catalase is a tetramer of four polypeptide chains, each over 500 amino acid long (Boon et al, 2001). The enzyme contains four ferric protoporphyrin groups per molecule (Molecular weight, 240,000)which corresponds to a protohaem content and an iron content (Fita et al, 2000).  The porphyrin heme group it contains allow it to react with the hydrogen peroxide.  The catalase activity of tissues varies greatly.  It is highest in the liver and kidney but low in connective tissues.  In connective tissues, it is mainly particle bound (in mitochondria and peroxisomes) where as it exists in a soluble state in enythrocytes.  The optimum pH for human catalase is ~ 7 (Machly and Chance, 1954).  The optimum temperature also varies by species (Tener et al, 2000).

1.12 HISTORY OF CATALASE

Catalase was first noticed as a substance in 1811 when Louis Jacques thenard, who discovered H2O2 (Hydrogen peroxide), suggested that its breakdown is caused by a substance.  In 1900, Oscar Loew, was the first to give it the name ‘Catalase’ and found its presence in many plants and animal tissues (Loew, 1900).  In 1937, catalase from beef liver was  crystallized by James B. Summer (Summer and Dounce, 1937) and the molecular weight worked out in 1938 (Summer and Gralen, 1938).  In 1969, the amino acid sequence of bovine catalase was worked out (Schroeder et al, 1969).

1.12.1 ACTIVITIES OF CATALASE

Catalase has been found to have a double function because it catalyses the following reactions:

1. Decomposition of hydrogen peroxide to give water and oxygen.

2H2O2­Catalase2H2O + O2

Hydrogen peroxide is a strong oxidizing agent and is potentially damaging to cells.  By preventing excessive accumulation of hydrogen peroxide, catalase allows important cellular processes which produce H2O2 as by-products (Martha et al, 1999).

3. Oxidation of hydrogen donors for example, methanol ethanol, formic acid and phenols with the consumption of one mole of peroxide:

ROOH + AH2 Catalase ROH + H2O + A.

Catalase falls into two main classes, the HPI and HPII catalase. HPII catalase catalyses the disproportionation of hydrogen peroxide while HPI carries out its double function.  HPI catalase exist as two isoforms: HPI – A and HPI – B and they sediment at slightly different densities (Vainshtein et al, 2000).

1.12.2  MOLECULAR MECHANISM OF CATALASE ACTION

While the complete mechanism of catalase is not currently known, the reaction is believed to occur in two stages:

H2O2 + Enz – Fe3+ = 0Enz+ - Fe4+ = 0  +  H2O

H2O2 + Enz – Fe3+ = 0Enz – Fe3+ = 0  +  H2O + O2

(Boon et al, 2001).

Here, Enz – Fe3+ represents the iron centre of the heme group attached to the enzyme.

As hydrogen peroxide enters the active site, it interacts with the amino acids Asn 147 and His 74 causing a proton to transfer between the oxygen atoms. The free oxygen atom coordinates, freeing the newly formed water molecule and Enz+ - Fe4+ = 0.  Enz+ - Fe4+ = 0 reacts with a second hydrogen peroxide molecule to reform Enz – Fe3+ and produce water and oxygen (Boon et al, 2001).  The reactivity of the iron centre may be improved by the presence of the phenolate ligand of Tyr 357 in the fifth iron ligand which can assist in the oxidation of the Fe3+ to Fe4+.  The efficiency of the reaction may also be improved by the interactions of His 74 and Asn 147 with reaction intermediates (Boon et al, 2001).  In general, the rate of the reaction can be determined by the micahelis – menten equation (Maass, 1998).

1.13 SUPEROXIDE DISMUTASE

Superoxide dismatase (SOD, EC 1.15.1.1) catalyse the dismutation of superoxide radical to hydrogen peroxide and oxygen (Fridovich, 1975).

02 + O-2 + 2H+ SuperoxideH1O2 + O2

      Dismatase

The dismutation of O2 to oxygen and H2O2 occur spontaneously but in the presence of the enzyme, the reaction is increased as much as 109 times as fast as the spontaneous rate superoxide Dismutase isoenzymes are compartmentalized in higher plants and play a major role in combating oxygen mediated toxicity (Fridovich, 1986).

1.13.1 TYPES OF SUPEROXIDE DISMUTASE

i. IRON SODS: This groups of Fe – SODs constitute the most ancient SOD group.  However, iron was the first metal used as a metal cofactor at the active site on the first SOD because of an abundance of iron in soluble Fe (II) form at that time Fe-SOD is found in prokaryotes and Eukaryotes.  In Eukaryotes, it was isolated from Euglena gracillis. In plant species, it is found in the chloroplast.  Fe – SOD is inactivated by hydrogen peroxide (Fridovich, 1975).

ii. Mn – SOD: Manganese SODs occur in the mitochondria and peroxisomes.  It carries only one metal atom per subunit.  Catalysis by Mn- SODs is through the attraction of negatively changed O2 molecules to a site formed from positively changed amino acid present at the active site of the enzyme.  The metal present in the active site donates an electron directly to the O2, reducing one )2 molecules, which in turns forms hydrogen peroxide by reacting with a proton.

iii. Cu-Zn – SOD:  Copper – Zinc SODs are found throughout the plant cell.  There are two different groups of copper-Zinc SODs.  The first group consists of cytoplasmic and periplasmic forms which are homodimeric. The second group comprises of the chloroplasmic and extracellular copper-zinc SODs which are homotetrameric.  The active site of each subunit functions independently when these subunits are separated and then coupled with an inactive subunit.  Newly formed enzymes shows full activity, producing evidence that the functional interactions between the subunits are not essential for full catalytic activity.

iv. Combialistic SODs: A combialistic iron/manganese SOD is an enzymatically active protein that can accommodate metal ligand. Some bacterial species can use a common SOD for both iron and manganese and utilize either of these two metals as the active metal cofactor.  According to the availability of the metal (Mamemasty et al, 1978.

In humans (as well as in other mammals), three gorms of superoxide dismutase are present.  SOD 1 is located in the cytoplasm, SOD2 is located in the mitochondria and SOD32 is found in extrracellular species.  SOD 1 is a dimmer while SOD2 and SOD3 are tetramers. SOD1 and SOD3 contain copper andzinc while cSOD2 contains manganeseg in its reactive centre.  The genes for SOD1, SOD2 and SOD3 are located on chromosomes 21, 6 and 4 respond 2 and SOD3 are located on chromosomes 21, 6 and 4 respectively.

1.13.2 PHYSIOLOGICAL IMPORTANCE OF SODs

The physiological importance of SODs is illustrated by the severe pathologies evident in mice genetically engineered to lack these enzymes.  Mice lacking SOD2 die several days after birth amidst massive oxidative stress (Li et al, 1995).  Mice lacking SOD1 develop a wide range of pathologies including hepatocellular carginoma (Elchuri et al, 2005), an acceleration of age-related muscle mass loss (Muller et al, 2006).  Mice lacking SOD3 do not show any obvious defects and exhibit a normal life span, though they are more sensitive to hyperoxic injury (Sentman et al, 2006).  In humans, mutaltions in SOD 1 can cause familial amyotrphic lateral sclevosis (Conwit, 2006).

1.13.3 USE OF SOD IN COSMETICS

Superoxide dismatase has been shown to be used in cosmetics products to reduce free radical damage to skin for example to reduce fibrosis following radiation for breast cancer (Campana et al, 2004).

1.14 PEROXIDASE

Peroxidase are a large family of enzymes.  A majority of peroxidase protein sequences can be found in the peroxidase database.  Peroxidase typically catalyse a reaction of the form.

ROOR1 + (2e-) + 2H+ ROH + R1OH

For many of these enzymes, the optimal substrate is hydrogen peroxide but others are more active with organic hydroperoxides such as lipid peroxides.  Peroxidases can contain heme cofactor in their active sites.  These are various kinds of peroxidase, which uses a wide variety of organic compounds as electron donors and acceptors.  Horse radish peroxidase has an accessible active site and many compound can reach the site of the reaction.

Glutathione peroxidase is a peroxidase found in human which contains selenocysteine. It uses glutathione as an electron donor and it is active with both hydrogen peroxide and organic hydroperoxide substrates.

1.14.1 ISOZYMES OF GLUTATHIONE PEROXIDASE

There are several isozymes encoded by different genes, which vary in cellular location and substrate specificity.  Glutathione peroxidase 1 (GPX1) is the most abundant version found in the cytoplasm of nearly all mammalian tissues, whose preferred substrate is hydrogen peroxide. Glutathione peroxidase 4 (GPX4) has a high preference for lipid hydroperoxides, it is expressed in nearly every mammalian cell, though at much lower level Glutathione peroxidase 2 (GPX2) is an intestinal and intracellular enzyme while Glutathione peroxidase 3 (GPX3) is extracellular especially abundant in plasma (Muller et al, 2001).

1.15 OXIDATIVE STRESS AND REACTIVE OXYGEN SPECIES

Reactive Oxygen species (ROS) intermediates are partially reduced forms of atmospheric oxygen (Halliwell and Gutteridge, 1999).  They typically result from the excitation of oxygen to form singlet or from the transfer of one, two or three electrons to oxygen to form respectively a superoxide radical hydrogen peroxide or hydroxyl radical (Das et al, 2000).

These radicals occurs transiently in aerobic organisms because they are also generated in plant cells during normal metabolic processes such as Respiration and Photosynthesis (Adada and Takahashi, 2000).  Although, some of them may function as important signalling molecules that alter gene expression and modulate the activity of specific defense proteins, although oxygen species can be extremely harmful to organisms at high concentrations.

Reactive oxygen species can oxidize protein, lipid and nucleic acids, often leading to alterations in cell structure and mutagenesis (Halliwell and Gutteridge, 1999).  There are many other potential sources of ROS in plant in addition to those that come from reactions involved in normal metabolism such as photosynthesis and respiration.  The balance between the steady state levels of different ROS are determined by the interplay between different ROS-producing and ROS-scavenging mechanisms and change drastically depending upon the physiological conditions of the plant and the integration of different environmental development and biochemical stimuli (Polle, 2001).

A variety of proteins function as scavengers of superoxide and hydrogen peroxide. These include among others superoxide dismatase, catalase, peroxidase (Muller, 2002).  These protein antioxidants are supplemented with a host of non-protein scavengers including: ascorbate and glutathione (Noctor and Foyer, 1998).

Antioxidant enzymes are chemical substances found in plants that act on free radicals.  Antioxidant enzymes work in several ways.  They may reduce the energy of the free radical, or give up some of their electrons for its use, thereby causing it to become stable.  They may also stop the free radicals from forming, they may also interrupt an oxidizing chain reaction to minimize the damage caused by free radicals.  The antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) serve as the cell’s primary line of defense in destroying free radicals.

1.16 OBJECTIVES OF THE STUDY

1. To investigate the toxic effect of nickel on sorghum root antioxidant enzymes.

2. To monitor the ameliorating effects of N.P.K. Fertilizer.

3. To monitor the level of lipid peroxidation.

.

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



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