Intracellular copper accumulation and biochemical changes in response to Cu induced oxidative stress in brassica species

Contents

1. Introduction

2. Review of Literature

3. Materials and Methods

4A Results (Part-1)

4B Results (Part-2)

4C Results (Part-3)

5. Discussion

6. Conclusion and Summary

References

ABOUT THE AUTHORS

Dr. R.C. Pantola obtained PhD in stress biology from Banasthali University, Rajasthan (India) and has over fifteen year experience in quality control, analytical research & development in chemistry and microbiology of the pharmaceutical industry. At present, Dr. Pantola is working as a Manager in the department of quality control, analytical research & development in chemistry and microbiology, Ind-swift Laboratories Ltd. Punjab, India. Dr. Pantola has published research and review articles in reputed journals related to pharmaceutical chemistry, microbiology and stress biology.

Dr. Afroz Alam obtained PhD in Botany from University of Lucknow, Lucknow (India) and has over sixteen-year research and teaching experience. At Present Dr. Alam is working as Associate Professor in Department of Bioscience and Biotechnology, Banasthali University, Rajasthan. Dr. Alam has published more than hundred research papers and review articles in the field of biological sciences with reputed journals. He has guided five students for their doctoral degree. He also has seven books to his credit. He is a life member of various scientific societies. He is also member of Editorial Boards of reputed international and national journals.

PREFACE

Out of 90 naturally occurring elements, 53 are considered as heavy metals, however, not all of them have biological significance. Heavy metals cannot be smashed, but can only be altered from one state to another. On the basis of their solubility in physiological conditions, 17 heavy metals are obtainable for living cells and of significance for the organism and ecosystem. Among these metals, Fe, Mn and Mo are important as micronutrients; Cu, Co, Cr, Ni, V, W and Zn are noxious elements with high or low importance as trace elements.

Most common heavy metals, namely, Cu, Cd and Zn create the major problem in contaminated soils that is completely different from organic pollutants. Unlike organic pollutants, these heavy metals cannot be biodegraded and therefore exist in the environment for extended periods of time. Hence, environmental pollution caused by these heavy metals becomes more frightening and tricky with ever increasing unplanned mining and unconstrained industrial activities. In present scenario of increasing industrialization, soil and water contamination is exceptionally alarming and widespread all over the developing world, including highly populated countries like China and India. Thus the toxicity of heavy metals in our environment is a worldwide problem and a growing hazard to the sustainable ecosystem. The present book is written on the basis of extensive research with the objectives to find out the uptake and toxicity of copper in three species of Brassica viz. B. juncea (L.) Czern., B. napus L. and B. rapa L. It also provides an insight regarding the tissue specific cellular buildup of copper in root, shoot and leaves of these species. The relationships with growth and biochemical changes under Cu induced stress are also discussed in this book.

ACKNOWLEDGEMENTS

The author acknowledges the help rendered by the physiologists from various parts of India for providing research articles related to heavy metal tolerance. Thanks are due to the Prof. Aditya Shastri, Vice Chancellor, Banasthali University, Rajasthan and Prof. Vinay Sharma, Dean, Faculty of Science and Technology and Head, Department of Bioscience and Biotechnology, Banasthali University, Rajasthan for providing basic facilities.

Dr. Gyan Singh Shekhawat, Associate Professor, Department of Botany, Jai Narain Vyas University, Jodhpur deserves special thanks for his support and encouragement during the course of present study.

Last but not least, thanks are due to our family and friends for their continuous support. We offer my genuine apologies for any inaccuracy and request suggestions from all the readers of this book to improve the publication in succeeding editions.

R.C. Pantola

Afroz Alam

ABOUT THE BOOK

Our knowledge of Cu and other heavy metals action inside plant cells is still elementary. A complete understanding of Cu tolerance and sensitivity in plants will be crucial for developing schemes either to genetically engineered plants that accumulate specific metals, either for the use in phytoremediation, or to improve human nutrition. This book would boost our understanding of the changes during Cu stress in Brasicca spp. It would be also useful to know about the complexity of the defense network, including antioxidants, against Cu stress, and this will be supportive of future research into developing salt-tolerant varieties.

Abbreviations

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1. Introduction

1.1 Heavy metals

Any metal or metalloid, which has definite environmental concern called heavy metal. The term created with indication of the detrimental effects of mercury, cadmium and lead, all of which are heavier than iron. Now this term applied to any other equally toxic metal. Frequently encountered heavy metals are arsenic, antimony, cadmium, copper, cobalt, chromium, lead, nickel, mercury, thallium, selenium, silver and zinc. Till date, there is no generally approved clarity of a heavy metal. Few parameters have been used to describe the term heavy metals such as atomic weight, atomic number, density, or their position in the periodic table. Most frequently, those metals and metalloids that have density criteria range from above 3.5 g/cm3 to above 7 g/cm3 are considered as heavy metals (Weast, 1984).

Out of 90 naturally occurring elements, 53 are considered as heavy metals, however, not all of them have biological significance (Weast, 1984). Heavy metals cannot be smashed, but can only be altered from one state to another (Marques et al., 2009). On the basis of their solubility in physiological conditions, 17 heavy metals are obtainable for living cells and of significance for the organism and ecosystem (Weast, 1984). Among these metals, Fe, Mn and Mo are important as micronutrients; Cu, Co, Cr, Ni, V, W and Zn are noxious elements with high or low importance as trace elements (Hu et al., 2015). Ag, As, Hg, Cd, Pb Sb, and U have not recognized functions as a nutrient and appear to be more or less toxic to plants and microorganisms (Godbold and Huttermann, 1985; Breckle, 1991; Nies, 1999).

Even if, a number of heavy metals are indispensable trace elements, some of them at elevated concentrations are toxic to all forms of life, including humans, animals, plants, and microbes. Heavy metals commonly apply an inhibitory action on microorganisms by blocking critical functional groups, shifting essential metal ions or amending the active conformations of biological molecules (Gadd and Griffiths, 1978; Wood and Wang, 1983). But, at comparatively low concentrations, a few heavy metal ions (Cu2+, Co2+, Ni2+ and Zn2+) are necessary for the microorganisms since they provide crucial cofactors for various metallo-proteins and enzymes (Tayyaba et al., 2014).

1.2 Heavy metals induced abiotic stress

Among 90 elements which are present in the earth’s shell, about 60 % are considered as heavy metals (Michalak, 2006). Some of them are essential for optimum ecosystem’s functioning while others are nonessential. On the basis of their solubility under physiological circumstances, 17 heavy metals are obtainable for living cells and important for the sustenance of living organisms and ecosystem as well. They are further classified on the basis of the concentration in ppm as macronutrients, trace and toxic elements such as Cr, Cu, Co, Ni, V, W and Zn are considered as toxic elements with high or low magnitude as trace elements. Ag, As, Cd, Hg, Pb, Sb and U have no identified functions as a nutrient and seem to be relatively noxious to living organisms (Sebastiani et al., 2004; Jami et al., 2010; Juknys et al., 2012). Essential metals are requisite by plants for their normal metabolism and growth, hence cannot be substituted by others in their biochemical role. For instance, Zinc (Zn) is not only an important part of a range of enzymes (peptidases, proteinases, dehydrogenases) but also involved in the metabolism of carbohydrates, proteins, phosphate, auxins, in RNA and ribosome formation in plants. Likewise, Copper (Cu) is needed for various vital physiological processes in plant’s metabolism (respiration, photosynthesis, nitrogen metabolism, carbohydrate distribution, cell wall metabolism, seed development) and also provide aid in disease resistance. (Fatnassi et al., 2013). The optimum level performing metabolisms from bacteria to humans is reliant on Cu and Zn (Adriano, 2001). Trace elements transform into toxic when their concentration goes beyond a precise threshold value (Sharma and Dietz, 2009; Yadav, 2010). In the environment, these metals exist in with an extensive array of oxidation states and coordination numbers which determine their specific properties.

Most common heavy metals, namely, Cu, Cd and Zn create the major problem in contaminated soils which is completely different from organic pollutants. Unlike organic pollutants, these heavy metals cannot be biodegraded and therefore exist in the environment for extended periods of time. Hence, environmental pollution caused by these heavy metals becomes more frightening and tricky with ever increasing unplanned mining and unconstrained industrial activities. In present scenario of increasing industrialization, soil and water contamination is exceptionally alarming and widespread all over the developing world, including highly populated countries like China and India (Cheng, 2003; Meharg, 2004). Thus the toxicity of heavy metals in our environment is a worldwide problem and a growing hazard to the sustainable ecosystem.

1.3 Sources of heavy metal contamination in soil, water and air

The atmosphere and underlying parent materials are considered as the two prevalent sources of heavy metal contamination in most of the terrestrial ecosystems (Parikh and Mazumder, 2015). Bedrock weathering and metal inputs from the environment are the prominent factors which determine the concentrations of heavy metals in soil. Volcanic activities and continental dusts are among the natural sources (Ernst, 1998). However, like any other environmental hazard, anthropogenic activities are the foremost cause of contamination. Man made activities like excessive use of metal based pesticides and metal enriched excreta in agriculture, the unrestrained use of mines and smelters, burning of fossil fuel, metallurgical engineering and electronics, military training, coal, natural gas, paper industries, etc. lead to the release of heavy metals, their subsequent accumulation and biomagnifications in ecosystems (Alloway, 1995; Ali et al., 2013). The use of rigorous agricultural practices, including supplementation of phosphate rich fertilizers, manure slush input and pesticide management is accountable for the shocking pollution of agricultural soils. Even though these practices not only augment considerably the crop yield by providing much needed defense to the plants, but also make available all the nutrients required for a speedy and improved growth. However, during the whole process, they also bring in large amounts of heavy metals (Cd, Cu, Pb and Zn) along with other organic noxious waste in soil which subsequently mounted up in the plant.

1.4 Regulatory guidelines for heavy metals

The guidelines are in place for control of metal contents in herbal and pharmaceutical products, generally employed as a process catalyst throughout the synthesis of pharmaceutically important materials (European Pharmacopoeia, 2014). Their application may show the way to residues in the final pharmaceutical material and accordingly in the final drug product. Such metal residues do not offer any therapeutic advantage to the patient and should therefore be estimated and controlled on the basis of safety and quality criteria. The instruction classifies metal residue into 3 classes based on their individual level of safety concerns. The limits are based on the maximal every day dosage, period of healing, and administration method of the drug products as well as the allowable daily exposure (PDE) of the metal residue (European Pharmacopoeia, 2014). The three categories are as below.

1.4.1 Category 1 Metals

This class includes those metals that are potential carcinogenic or likely a causal agents of any other important toxicity.

1.4.2 Category 2 metals

This class includes metal with lesser noxious potential to humans. They may be trace elements that are necessary for dietary purposes or they are frequently found in food stuff or commonly accessible dietary supplements.

1.4.3 Category 3 metals

This group includes metals that have least safety worry. They don’t have any significant toxicity. Their safety report is well recognized. They are usually well accepted up to dose normally available with the direction of medicinal products. Usually, they are omnipresent in the ecosystem.

Table 1.1: Class, exposure and concentration limits of individual metals for Drug Products (European Pharmacopoeia, 2014)

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Table 1.2: Limits of copper for foodstuffs (FSSAI, 2011)

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1.5 Brassica species: a model plant for study, known as a heavy metal accumulator

The members of the family Brassicaceae are outstanding for their hyperaccumulating capability of the heavy metals (Broadley et al., 2001). Owing to this fact, momentous consideration has been paid to the various taxa of this family. The plants of this family are well known for having great biomass and ability to build up high magnitudes of heavy metals (Dushenkov et al., 1995; Castiglione et al., 2007; Ahn et al., 2012). According to Kumar et al. (1995), Brassica have higher growth rates and they have an excellent ability to bear and build up the metals. Species like, Brassica juncea (L.) Czern., Brassica nigra Koch, Brassica campestris L., Brassica napus L., and Brassica oleracea L. of the family Brassicaceae show somewhat equal capability to uptake and build up alongwith subsequent transport the heavy metals towards their stem. Although, almost all of the examined crops of the family Brassicaceae do mount up the metals in their tisues, but, B. juncea showed the most excellent capability to accumulate and transport Cd, Cr, Cu, Ni, Pb, and Zn towards their aerial parts (Boye, 2002; Anjum et al., 2008).

In this study, potential of Brassica species was sought to analyze in the presence of elevated levels of copper. The element selected for this study is essential for various physiological processes, over accumulation of which can lead to deleterious effects. It is assumed that enhanced supply of cations could lead to increased uptake and accumulation by this plant, as it has been shown to display this propensity. The study was carried out using Brassica juncea (L.) Czern., Brassica napus L. and Brassica rapa L. grown hydroponically and its response to elevated levels of the copper, shall be monitored in relation to its capacity for accumulation.

1.6 Need and importance of the present study with the objectives

Family Brassicaceae becomes very important family whenever heavy metal accumulation is concerned several species are hyper accumulator more than one metal (Prasad and Freitas, 2003; Qadir et al., 2004). Brassica species are very significant oil crops. Mustard oil is one of the major cooked oils in India. This oil has also acquired therapeutic significance. The remains of the seeds are also used as fertilizer and for feeding cattles. Indian mustard (B. juncea) is a rapidly growing plant species that produces a superior biomass even in heavy metal contaminated soils. Since most of the identified hyper-accumulator species are sluggish in growth and produce low biomass, there is an acute need to identify those plants that should have high expression of those genes which are associated with the accumulation of heavy metals, that results in high biomass. This can be a promising approach for developing plants that can be used in phytoextraction technology. Brassica species can be proved useful for studies related to hyper-accumulation of heavy metals and their tolerance mechanisms. For Instance, cellular detoxification mechanisms under Cu stress likely to be useful in the upcoming research, especially for genes concerned in metal tolerance and accumulation. Such genes can be engineered to other members of this family and evaluated for their heavy metal restraint, buildup and phytoremediation impendings. Upshot of such studies may lead to a consideration of plant-metal interactions, cellular metabolic processes involved in detoxification of ROS generated by the metals and tolerance to heavy metal in plants that will result in vital relevances of concentrated ecological acceptable effort.

2. Review of Literature

Metal pollution in the environment is a major problem. Occurrence of metals is one of the most prevailing forms of pollution observed at throw away sites and their removal from contaminated soils is among the most technically difficult tasks. Elemental pollution causes health problems in livestock and human beings. The productivity of agricultural land and soil quality needs improvement in contamination of zinc, iron, copper and potassium (Kalpana et al., 2012).

2.1 Brassica species

The herbaceous oilseed crop Brassicas belong to a family Brassicaceae (Cruciferae). These oilseed crops are grown in the temperate agricultural zones of the world. The ability to survive and produce seeds under comparatively harsh conditions has made them adaptable to cultivate in cool climate, high elevations and subtropical condition. Four closely related Brassicaceae species (B. carinata, B. juncea, B. napus and B. rapa) are grown all over the world for oil production (Downey and Robbelen, 1989). B. juncea has relatively older record as a domesticated plant than B. napus, and the species may be an interspecific cross between B. rapa and B. nigra (Uchimiya and Wildman, 1978). The ideal plant for phytoextraction grow rapidly, produces high amount of biomass and tolerate and accumulate high metal concentrations are also belongs to Brassicaceae (Jing et al., 2007).

2.2 Properties of Brassicaceae family

The Brassicaceae family contains a number of agriculturally important species that are grown around the world as oil seeds, vegetables and for production of condiments (Snowdon et al., 2007). According to seed coat color two market classes of B. juncea exist, brown mustard has dark brown seed coat and oriental mustard has golden yellow seed coat. Both seeds are small in size (about 1.6 mm in diameter) and the seed coat contains low amount of mucilage (Kimber and McGregor, 1995; Auger et al., 2010) compared to Sinapis alba. Allyl glucosinolates is the main flavor precursor of B. juncea seed and the oil extracted air dried meal contains 150-200 μmol g-1 of allyl glucosinolate (sinigrin). Sinigrin is non volatile and the activity of myrosinase enzyme produces an aglycone that can decompose to a number of compounds including allyl isothiocyanate contributing to hot pungent flavor of mustard. The crude oil and protein contents of B. juncea seed is about 33-45% and 23-29% respectively. The content of seed oil and protein is affected by environmental conditions (Uppstrom, 1995: Maria et al., 2011). Predominant unsaturated fatty acids of B. juncea oils are oleic acid (about 20%), linoleic acid (about 10-15%) and linolenic acid. The oil free meal of B. juncea is a good source of protein (Maria et al., 2011).

2.3 Copper level in the environment

The precise annual quantity of copper inflowing into the overall ecosystem is not well-known, but its predictable range is 211,000 metric tons -1.8 million metric tons (NAS, 1977). Approximately 80.7% of this high amount of copper is laid down in terrestrial partition, 15.7% is released in the hydrosphere, and 3.6% goes into the atmosphere. In case of soils, it may be preserved for about 1,000 years; in the hydrosphere, copper may exist in the deep marine system for about 1,500 years; whereas, in the air, it remains for about 13 to 15 days. About 73% of atmospheric copper is mainly a consequences of anthropogenic activities like production of copper and and oblivious burning of fossil fuels (0.1–6.4x106 kg/year), the rest is comes from natural sources that comprise volcanogenic atoms (0.9–18x106 kg/year), windblown dirts (mean worldwide emission of 0.9–15x106 kg/year), sea salt sprays (0.2–6.9x106 kg/year), and forest fire (0.1–7.5x106 kg/year), rotting vegetation also contribute to some extent (Nriagu, 1979; WHO, 1998; Ismail, 2014).

On the basis of given data, approximately 1.3–38.8x106 kg/year copper emission takes place in the atmosphere. Anthropogenic activities are chiefly responsible for this extent of discharge of copper. These activities are mostly ignorant, which include production of nonferrous metal, iron and steel production, combustion of coal, oil and gasoline, wood production, waste incineration, industrial applications, non-ferrous metal mining, and use of phosphate fertilizer. According to an estimate, merely 0.04% of environmental copper are released into the air (Perwak et al., 1980). Atmospheric anthropogenic emitted copper has been estimated 35x106 kg/year,while natural emissions of copper were estimated 28x106 kg/year (Nriagu and Pacyna, 1988; Nriagu, 1989; Giusti et al., 1993). Various sources contribute to this extent of anthropogenic and natural emissions of copper in different range, which are shown in Tables 2.1 and 2.2.

Table 2.1: Copper Emissions from Natural Sources (x106 kg/year)

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Table 2.2: Copper Emissions through Anthropogenic Activities (x106 kg/year)

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2.4 Heavy metal avoidance, tolerance and resistance mechanisms in plants

To combat with heavy metal stress, several plants have evolved tolerant races with abilities to live and flourish on such metalliferous soils. They have adapted well through certain adjustment in key mechanisms which are involved in the common homeostasis and constitutive tolerance to essential metal ions that are found in plants. At the cellular level a great array of potential mechanisms are now known that might be involved in the detoxification of these metals and consequently stress tolerance against heavy metals (Hall, 2002). Primarily these all seem to be involved in shunning build up of toxic concentrations at susceptible sites inside the cell and therefore averting the destructive effects, rather than developing the resistant proteins against the heavy metal consequences. Thus, it has been revealed that frequently the tolerant plant species exhibit better averting and homeostatic mechanisms to avoid the inception of stress. Conversely, there is modest evidence regarding tolerant species or ecotypes which show an improved oxidative defense mechanism (Dietz et al., 1999; Anjum et al., 2011).

The approach to avoid upsurge of heavy metal are varied. They include the roles of mycorrhizas and extracellular exudates as well. Tolerance could also engage the plasma membrane, either by dropping the uptake of heavy metals that have come into the cytosol. A variety of potential mechanisms also exist within the protoplast for instance, the reinstatement of damaged proteins due to stress involves heat shock proteins (HSPs) or metalothionines and for the chelation of metals by amino acids or peptides and organic acids, or by keeping away from metabolic processes by transport into the vacuoles.

2.4.1 Mycorrizhas

The strategies are diverse for shunning toxicity caused by heavy metal toxicity. One such extracellular strategy includes Mycorrhizas to avoid metal toxicity (Marschner, 1995; Jentshcke and Godbold, 2000). Though, very few studies have been done in past which produced straight confirmation of the mitigation of metal toxicity by micorrhizal (fungal) association (Leyval et al., 1997; Jentshcke and Godbold, 2000; Schutzendubel and Polle, 2002). The studied heavy metals were Zn (Brown and Wilkins, 1985), Ni (Jones and Hutchinson, 1986), Al (Schier and Mc Quattie, 1996) and Cd (Jentschke et al., 1999). In Recently, in case of Paxillus involotus, detoxification of Cd involved Cd binding to the vacuole (Blaudez et al., 2000; Yang and Chu, 2011).

2.4.2 Cell wall and plasma membrane

As a first line of defense in opposition to heavy metals, roots secrete exudates into the contiguous soil matrix. One of the major tasks of root exudates is to chelate metals and to avoid their uptake in the interior of the cells (Marschner, 1995). Salt et al. (2000) studied that Ni-chelating histidine and citrate are exist in root exudates which diminish the uptake of Ni from the top soil. In the apoplastic zone the binding of Cu and Zn also helps to control the metal content of root cells (Dietz, 1996). On the cell wall of root cells distinct cation binding sites exist that influences the availability of ions of uptake and their subsequent diffusion into the apoplast by permitting the metal exchange (Suzuki et al., 2012). In the immobilization of toxic heavy metal ions, the cell wall can also play a critical function in the prevention of unwanted heavy metal uptake by providing hystidyl groups and pectic sites, and extracellular carbohydrates for instance, callose and mucilage. Wang et al. (1992) showed that diverse tobacco genotypes with chemically divergent root cell wall surfaces have diverse sensitivities to Mn toxicity.

2.4.3 Chelation or Vacuolar compartmentalization

Inside the cell, heavy metal ions that are not immediately required metabolically may reach toxic concentrations, and plant cells have evolved various mechanisms to store excess metals to prevent their participation in unwanted toxic reactions. If the toxic metal concentration exceeds a certain threshold inside the cells, an active metabolic process contributes to the production of chelating compounds. Specific peptides for instance, metallothioneins (MTs) and phytochelatins (PCs) are used to chelate metals inside cytosol and to sequester them in specific subcellular compartments. A large number of small molecules are also involved in metal chelation inside the cells, including organic acids, amino acids, and phosphate derivatives (Rauser, 1999; Tikkanen and Aro, 2012).

2.4.4 Metallothioneins and phytochelatins

The two well known heavy metal binding polypeptides include the small gene encoded, cysteine polypeptide-metallothioneins (MTs), and the enzymatically synthesized; cysteine rich peptides- phytochelatins (PCs) (Coldsbrough, 2010). Phytochelatins are diminutive metal binding peptides having a construction: (g-glu-cys)n-gly, (g-glu-cys)n-glu etc. (Dalcorso et al., 2010). While, the tripeptide glutathione (GSH, γ-Glu-Cys-Gly) is a substrate for the biosynthesis PCs. Phytochelatins synthesis from GSH is catalyzed by a transpeptidase explicitly, phytochelatin synthase (Klapheek et al., 1995; Choudhary et al., 2011). Phytochelatin synthase erstwhile revealed to be activated only in the existence of heavy metal ions, in particular Ag, Au, As, Cd, Cu, Hg, Pb, Sn and Zn (Cobbett, 2000). In case of Brassica juncea, over-expression of glutathione synthase enhances PCs synthesis and Cd tolerance as well (Peleg and Blumwald, 2011; Ha et al., 2012).

2.4.5 Cellular antioxidant defense

Antioxidant defense systems keep the customarily formed ROS at low intensity and stop them beyond the toxic verges. Abiotic stresses disturb the balance between ROS production and detoxification (Sharma and Dietz, 2009). The antioxidant association consists of enzymatic and non-enzymatic mechanisms, for instance, O2˙¯ hunting by superoxide dismutase (SOD) and H2O2 breakdown by ascorbate peroxidasse (APX), peroxiredoxins (PRXes) and catalase (CAT) are principally connected with the upholding the steady state of cellular redox. Isoforms of APX, SOD and PRX are restricted in numerous subcellular compartments, whereas, catalase is generally contained in peroxisomes. They sturdily vary in their substrate attractions and make sure a stiff control over H2O2 concentrations and restrict it to very low levels. Enzymes like, glutathione peroxidase (GPX) and glutathione-S-transferases (GSTs) also play an important role to perfect balance of cellular redox. In heavy metal stressed plants, activities of antioxidant enzymes reveal stimulation, refusal effect and containment depending on the plant species, metal ion, concentration and duration of exposure (Hossain and Fujita, 2010).

2.5 Heavy metal effects on antioxidant activity

Toxicity caused by heavy metal is connected with the augment in the activity of many enzymes for instance, peroxidase and glucose-6-phosphate dehydrogenase in the foliage of plants grown in metal contaminated soil (Yruela, 2005). Being essential cofactors of most antioxidant enzymes, these metal ions have an imperative role in the antioxidant network. They are concerned with the direct or indirect production of reactive oxygen species (ROS) and other free radicals. The ROS produced in leaf cells are scavenged by the complex action of enzymes like superoxide disumutase (SOD), ascorbate peroxidases (APX), catalase (CAT), glutathione peroxidases (GPX) and glutathione reductases (GR) that are involved in antioxidant coordinations. Proline is recognized very well for its significant role in the detoxification process of active oxygen in Cajanus cajan and Brassica juncea under the stress condition caused by heavy metals (Yruela 2005; Yruela, 2009; Burkhead et al., 2009).

2.6 Heavy metal induced oxidative stress and stress tolerance

Different abiotic stresses as drought, salt, and cold may cause common effects on the growth and development of plant by disrupting the osmotic potential that imbalance the cellular water equilibrium causing osmotic stress with subsequent production of stress hormone i.e. abscisic acid (ABA) for osmotic tuning (Wang et al., 2003). ABA is the most prominent endogenous messenger in response to stress, and therefore the pathway of ABA signaling is essentially involved in various adaptive routes (Tuteja, 2007). Under abiotic stresses, osmotic stress and the ubiquitous oxidative stress, the intricate network of abiotic stress related signaling pathways have been comprehensively studied and several evidences are put forward for various steps or levels to a greater extent. It has been proven that the undesirable ROS overproduction is the major consequence of heavy metal stress that causes irreversible cellular damages (Miller et al., 2008).

Reactive oxygen species have been re-evaluated in recent past as major signal molecules in the regulation of usual cellular function and subsequent growth (Rhee, 2006). In plants, the intricate and well-organized network of hunting methods permitted conquering toxicity of ROS. Chiefly, sensing of toxicity is done by the hydrogen peroxide (H2O2) that is formed by cytosolic membrane bound NADPH oxidases, and it serves as a signal in a large array of abiotic stress linked responses (Bailey-Serres and Mittler, 2006; Mittler et al., 2004).

2.7 ROS as a signaling molecule

Although ROS are usually known for their toxicity related concerns but their role also has been assessed as key signaling molecules in regulation of various vital cellular utilities and development (Rhee, 2006). In stress tolerant plant species, the intricate and well-organized system of hunting methods allowed conquering ROS toxicity and using a few of these noxious molecules, for instance, hydrogen peroxide (H2O2) formed by cytosolic membrane bound NADPH oxidases, serves as a signal molecule in a many of the responses related to abiotic stress (Bailey-Serres and Mittler, 2006; Mittler et al., 2004; Neill et al., 2002). In cells exacting sets of genes have been activated in response to buildup of ROS, several of which are considered as central regulators of stress related responses, for instance, zinc finger protein, WRKY transcription factors and heat shock proteins, Rboh genes, and multi-protein bridging factor 1c (Miller et al., 2008).

2.8 Other sources of ROS generation in plants

Detoxification reactions that occur in endoplasmic reticulum and cytoplasm, catalyzed by cytochrome P450 have gained considerable attention as additional imperative sources of ROS production in plants (Garg and Manchanda, 2009). At the level of plasma membrane and apoplastic zone production of ROS has also been reported. In apoplast of plant cells, amine oxidase and oxalate oxidase has been considered as potent source H2O2, however, these cell wall peroxidases of cell wall are pH dependent (Tuteja et al., 2009). Likewise, alkalization of apoplast in response to elicitor identification leads the oxidative explode with remarkable generation of H2O2 during stress (Bolwell and Woftastek, 1997; Russo et al., 2007).

2.9 ROS scavenging antioxidant defense mechanism

Introduction of plants to adverse environmental settings for instance, very high temperature, elevated levels of heavy metals, severe drought, limited irrigation, atmospheric contaminants, nutrient insufficiency, or saline stress can augment the creation of reactive oxygen species (1O2, O2.-, OH. and H2O2). Under ROS build up situation for protection purposes, plant cells and its organelles like mitochondria, chloroplast and peroxisomes employ antioxidant defense systems for their protection. Several research findings had recognized that the stimulation of the cellular antioxidant mechanism is imperative for safety against various stresses, (Tuteja, 2007; Khan and Singh, 2008; Singh et al., 2008a). The major parts of antioxidant defense system are non enzymatic and enzymatic antioxidants. Enzymatic antioxidants include APX, CAT, SOD, DHAR, GR and MDHAR while non enzymatic antioxidants are AA (water soluble), carotenoids, GSH, and tocopherols (lipid soluble) (Mittler et al., 2004; Singh et al., 2008a).

2.10 ROS scavenging enzymatic antioxidants

Superoxide dismutase (SOD), the omnipresent enzyme in all aerobic living beings and in all subcellular compartments, considered as the most efficient intracellular enzymatic antioxidant that is somewhat susceptible to ROS mediated oxidative stress. In case of two cultivars of Brassica compestris under Cu stress, Li et al. (2009) observed a significant increase in the activity of SOD. Another ROS linked enzyme i.e. tetrameric heme containing Catalase (CAT) has the capacity to dismutate H2O2 into H2O and O2 directly, and is found essential for ROS detoxification under stressed circumstances (Garg and Manchanda, 2009). The role of Ascorbate peroxidase (APX) is also indispensible in protecting cells of plants and other organisms by rapid scavenging of ROS. Arvind and Prasad (2003) have reported an increased leaf APX activity under Cd stress in Ceratophyllum demersum. Similar observation was revealed in case of Brassica juncea by Mobin and Khan (2007). The other ROS scavenging enzymatic antioxidants are guaicol peroxidase (GPOX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), monodelhyde ascorbate reductase (MDHAR), glutathione S- transferase(GST) and glutathione peroxidase (GPX) that have crucial role in ROS scavenging mechanisms (Ahammed et al ., 2013).

2.11 Copper toxicity

Copper (Cu) is considered as one of the indispensable trace elements because its requirement as a cofactor for many enzymes and other cellular activities like uptake of iron (Jambunathan 2010; Kim et al., 2011). In case of enzymes, namely Cu/Zn-superoxide dismutase (Cu/ZnSOD), cytochrome C oxidase, ascorbate oxidase, amino oxidase, laccase, plastocyanin and polyphenol oxidase are good examples. While, at the cellular level, the function of Cu is essential in cell wall metabolism, cell signalling, oxidative phosphorylation, mobilization of iron and the molybdenum cofactor biogenesis (Yruela, 2005; Pilon et al., 2006; Puig et al., 2007). Though, higher concentration of copper is lethal and also known as potentially carcinogenic agent. Neuro-degenerative problems like Alzheimer’s disease and autism are few other examples that have been associated with exposure of copper in humans (Rotilio et al., 2000; Kawanishi et al., 2002). Through various studies, it has been somewhat established fact that copper applies its effects during induction of oxidative stress at the cellular level as well (Avery et al., 2001).

In all living systems Cu is an essential redox-active transition metal. In a living organism, it has a capability to exist in multiple oxidation states (Cu2+and Cu+) and because of this reason it participates in many physiological processes. In histidine side chains, Cu2+ is regularly bound by nitrogen, whereas Cu+ have a preferred interaction with the sulfur in cysteine or methionine. For some metalloproteins Cu acts as a structural element. Several of which are concerned in choloplastic and mitochondrial electron transport in response to oxidative stress (Das et al., 2015).

Therefore, plants necessitate the existence of Cu in vivo for usual development and growth, and in deficiency of Cu, plants develop precise deficiency symptoms, most of which influence juvenile leaves and gametohytic organs. Conversely, the redox properties that make Cu indispensable element also contribute to its intrinsic toxicity. However redox cycling involving Cu2+ and Cu+ forms can catalyze the oxidative burst of extremely toxic hydroxyl radicals, which cause serious injuries to cells, usually involves damages to membrane bound lipids, cell membranes, nucleic acids, proteins and several other biomolecules (Halliwell and Gutteridge, 1984). Even though Cu generally binds to proteins, but it also has ability to begin oxidative burst and also hinder some of the important cellular processes, for instance pigment synthesis, photosynthesis, permeability of the plasma membrane and other metabolic mechanisms, creating an influential inhibition in the different stages of plant development (Kupper et al., 2003; Bertrand and Poirier, 2005; Yruela, 2005). Toxicity may result from several factors, for instance, binding to sulfhydryl groups in proteins, thus inhibiting protein function or enzymatic activity, causing a shortage of other essential ions in living systems, spoiled cell transportation mechanisms and oxidative damage. The toxic symptoms of Cu excess include necrosis, chlorosis, stunting, and inhibition apical growth. Surplus Cu concentration in cells can inactivate and interrupt protein structure as a result of inevitable binding to proteins.

2.12 Uptake and toxicity of copper in plants

Overloaded heavy metal content in polluted soil coupled with decrease in crop production is a worldwide problem because it inhibits the plant growth to a great extent. Several procedures were tried in past using metal accruing plant species which are able to remove these disproportionate metals from terrestrial as well as aquatic systems (Salt et al., 1995). Now, it has been almost established that Brassica juncea can be used as a model plant species in the reclamation of heavy metal polluted soils (Muhammad, 2010). The biggest advantage of using this particular species is a high biomass production that is needed to accumulate elevated amount of heavy metals such as Cu, Cd, Ni, Pb, and Zn (Salt et al., 1995a).

Above optimum concentration copper was reported to inhibit usual growth of the plants by interfering with vital metabolic processes (respiration and photosynthesis) of the cell (Dubey, 2011; Villiers et al., 2011). In Cu rich soil, the grown-up plants exhibit decreased biomass and symptoms of chlorosis (Wahida et al., 2013). Under such conditions, leaves show poorer chlorophyll content and changed structure of chloroplast along with altered composition of thylakoid membrane (Dalcarso et al., 2010; Carrier et al., 2003; Quartacci et al., 2000). Generally, degradation of grana stacking and stromal lamellae, augment in the quantity and size of plastoglobuli, and manifestation of intra-thylakoidal inclusions were experimentally proven. It was observed that Cu hampers the biosynthesis of the photosynthetic apparatus by altering the protein and pigment symphony of photosynthetic membranes (Maksymiec et al., 1994; Gamalero et al., 2009). In addition, unusual peroxidations of lipids reduce of lipid content and subsequently change the fatty acid composition of thylakoid membranes (Maksymiec et al., 1994; Sharma and Dubey, 2007). As an outcome of such alterations, abnormal adjustment in the membrane fluidity of photo system II (PS II) was observed (Quartacci et al., 2000). Conversely, the decrease in the photochemical activity due to Cu stress is associated in vivo by a modification in the organization and composition of the thylakoid membranes that can exert a controlling effect on the conformation and function of the photo systems I and II (Lidon et al., 1993; Gamalero et al., 2009). Baszynski and Kruppa (1995) proposed toxic level of Cu is capable of inducing those processes that engage either in the obliteration of the polypeptide composition in oxygen evolving complex or they interact with ions required for proper functioning of the complex for instance Mn, Ca and Cl.

Cu is known to catalyze the creation of hydroxyl radicals (OH.) through Haber-Weiss reaction by a non enzymatic chemical reaction among H2O2 and superoxide (O2.-) (Halliwell and Gutteridge, 1984). Therefore, the surplus existence of Cu can originate oxidative stress in plants with consequent augment in the antioxidant responses because of amplified production of extremely toxic free radicals of oxygen. It was studied that surplus Cu content in plants caused oxidative stress stimulating alterations in the activity and amount of some components of the antioxidative pathways explicitly ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase, guiacol peroxidase, monodehydroascorbate reductase and superoxide dismutases (Wang et al., 2004; Hossain et al., 2009; Hossain et al., 2010). Several antioxidant responses were experimentally reported in roots and leaves which are dependent on concentrations level of Cu and time. Drazkiewicz et al. (2003) reported that the ascorbate-glutathione cycle has also been involved in response to excess Cu content.

2.13 Tolerance mechanisms to copper toxicity

In plant species that are able to grow in polluted soils that have elevated concentrations of heavy metals, tolerance could be attained at cellular level by a variety of impending mechanisms that might be concerned in detoxification. Primarily, these methods appear to be involved in shunning the buildup of toxic concentrations at responsive sites inside the cell averting the harmful effects more willingly than mounting resistant proteins that can defend against the effects of heavy metal. The probable cellular methods involved in stress tolerance include (1) decline in metal content either through the extracellular exudates or by limiting the uptake by mycorrhizal association, (2) rushing of the efflux pumping of the heavy metals at the level plasma membrane, ( 3) metal chelation through metallothioneins, phytochelatins, heat shock proteins and organic acids, (4) vacuolar compartmentation of heavy metals (Hall, 2002; Singla et al., 2006). There is a modest verification regarding an enhanced oxidative defense of tolerant species or ecotypes; to a certain extent, tolerant species demonstrate better prevention and homeostatic mechanisms to avert the stress conditions (Dietz et al., 1999; Sharma and Dietz, 2009).

2.14 Biochemical changes in detoxification of copper

Intracellular biochemical damage activates reduced forms of oxygen. These free radicals of oxygen are generated within the cells, when molecular oxygen receives electrons from any other molecules and several intracellular reactions reduce oxygen to hydrogen peroxide (H2O2) or superoxide. Though, these molecules are not extremely reactive but they can produce hydroxyl radicals, which are supposed to be responsible for many processes of the oxidative injuries in living systems (Halliwell and Cutteridge, 1990; Yruela, 2005). At the cellular level, plants have a great range of impending mechanisms that may be concerned in the detoxification process of stress caused by heavy metal and as a result exhibit tolerance against the heavy metal stress (Hall, 2002; Lai et al., 2010).

2.15 Accumulation of metal by Brassica species

Family Brassicaceae has Brassica species that are now considered as one of the best hyper-accumulators. However, earlier, it was thought that Brassica species that have great power of hyper-accumulation of hazardous heavy metals were not feasible for the process of phytoextraction. Consequently, researchers were looking for any other high biomass producing members of the Brassicaceae which accumulate large quantities of toxic metals (Dushenkov et al., 1995; Komarek et al., 2007). Later on, workers like Kumar et al. (1995) evaluated many rapidly growing species of Brassica for their capacity to withstand high concentration of metals and subsequent accumulation in vegetative parts.

Abbildung in dieser Leseprobe nicht enthalten

Table 2.3: Possible species of Brassica for the phytoextraction of different metals

Abbildung in dieser Leseprobe nicht enthalten

Brassica juncea, B. nigra, B. campestris, B. napus, and B. oleracea were assessed for these purposes. Although, all Brassicas have the ability to buildup metal, but B. juncea showed best ability to buildup excess amount of metals (Cu, Cr, Cd, Pb, Ni, and Zn) and their subsequent translocation to different parts of the plant (Table 2.1). Brassica juncea (Indian mustard), an oil seed crop, is the well recognized plant species for phytoremediation purposes. Its genetic make-up and biochemical characteristics support the hyperaccumulation of heavy metals much better in comparison to any other species (Ibrahim et al., 2009).

This crop produces high biomass even in heavy metal contaminated sites. Possible mechanism involved in metal accumulation is the uptake of metals in the root via solubilizing the metal from soil matrix and transported to the leaves where it is detoxified or chelated and finally sequestered and volatilized (Mukhopadhyay and Maiti, 2010). Thus, Indian mustard has great potential to uptake excess amount of metals such as Cd, Cu, Ni, Pb, Se and Zn from the contaminated sites and their subsequent buildup to a very high level in vegetative parts (Marzena et al., 2011; Kafeel et al., 2014).

2.16 Role of chelators for accumulation of metals in Brassicas

There are various methods for augmenting the accumulation characteristics of different members of the family Brassicaceae. The foremost method concerned with the strengthening of soil by addition of such compounds that are able to hasten heavy metal uptake. Several compounds are now recognized which can serve as possible chelators of heavy metals (Table 2.4). However, several drawbacks are also known related to their usage including possible toxicity with lesser biomass production and environmental related hazards due to uncontrolled movement of these heavy metals that can affect the normal course of our food chain.

Abbildung in dieser Leseprobe nicht enthalten

2.17 Uses of plants for phytoremediation

Use of plants to remove sequesters or to detoxify pollutants through chemical, physical, and biological processes is known as phytoremediation (Cunningham and Ow, 1996; Hu et al., 2013). The effectiveness of phytoremediation depends on the proper management and sustenance of crucial plant species that should have adequate shoot and root biomass growth and particular activities that can prop up a thriving microbial conglomerate which is supplementary for phytoremediation in the rhizosphere. For a feasible and effective phytoremediation, it is essential to find rapid growing, hyperaccumulating and metal tolerant plant species with a robust root system that can produce abundant above ground biomass. On the above parameters, Baker (1981) classified plants into three major groups. He termed ‘excluders’ to those plants which have the ability to grow in metal contaminated soil and keep unrestricted root-to-shoot transport by maintaining the shoot concentration at low level up to a critical soil value. Likewise, accumulators are those plants that accumulate the higher levels of heavy metals in their aerial part, and the indicators, which can uptake metals and subsequently transfer them to the shoot. The process is regulated in such an order that internal concentration reflects external levels, at least until toxicity take place. Several biochemical reactions occur in heavy metal stressed plants. Many of these reactions are created by the dislocation of protein cationic centers or to augment of the reactive oxygen species (ROS). The plant species that have superior capabilities to fiddle with the toxicity effects of heavy metals are competent to incessant exist in heavy metal polluted soils and hence they are preferred contenders for phytoremediation point of view (Violante et al., 2010).

The phytoremediation process can be categorized in five major subgroups, essentially depending on the plants used for the remediation purposes (i) The method in which plants are utilized to concentrate metals uptake from the soil into the roots and aerial parts of the plant refers as Phytoextraction, (ii) The utilization of metabolic potential of plants and microorganism of rhizosphere zone to decompose organic pollutants is termed as phytodegradation or rhizodegradation, (iii) The utilization of roots of plants to absorb and concentrate metals from effluents is termed as rhizofilteration, (iv) The suppression process using plants and their allied microbes to mechanically calm down the site and diminish the movement of heavy metals through absorption and precipitation by plants, which reduces their bioavailability and subsequent pollutant transfer to other ecosystem compartments and the food chain is known as phytostabilisation, (v) Phytovolatilisation or rhizovolatilisation is a removal mechanism which employs metabolic capabilities of plants and allied rhizosphere microorganisms to change pollutants into volatile materials that are released to the atmosphere (Chaudhry et al., 1998; Khan, 2005). However, phytoextraction and phytostabilization are two most relevant methods used frequently for remediation of metal contaminated soil (Garbisu et al., 2002; Epelde, 2010).

2.17.1 Genetically engineered plants for phytoremediation

Selected plant species for phytoremediation possibly genetically engineered to improve their toleration and accumulation capabilities for heavy metals by over expression of those genes which encode enzymes concerned in heavy metal tolerance and accumulation (Clemens, 2001; Clemens and Kramer, 2002; Pilon-Smits and Pilon, 2002).

To combat with stress induced by heavy metals, plants have developed several mechanisms. For instance, synthesis of the phytochelatins (PCs) and S-rich metal chelators glutathione (GSH) (Hall, 2002; Gasic and Korban, 2007). GSH, the most copious low molecular weight thiol compound that exists in plants (Bergmann and Rennenberg, 1993; Hell, 1997), its synthesis occurs in the course of a two step ATP dependent enzymatic pathway (Noctor et al., 2002). As a foremost reaction, γ -glutamylcysteine (γ -EC) is formed from glutamate and cysteine by the activity of γ -glutamylcysteine synthetase (γ -ECS) (Hell and Bergmann, 1990), γ -ECS is encoded by the gshI gene (May and Leaver, 1994). In next step, S-rich metal chelators glutathione (GSH) is created by the splicing of γ -EC and glycine in the reaction that is catalyzed by gshII gene encoded glutathione synthetase (GS) (Wang and Oliver, 1996).

In the protection mechanisms of plants against various environmental stresses like oxidative tress, heavy metals toxicity and xenobiotics, glutathione plays a fundamental task (Huang et al., 2010 ; Foyer and Noctor, 2005; Freeman et al., 2005). Undeniably, GSH works as an antioxidant, extinguishing the reactive oxygen species (ROS) produced in reply to stress before these reactive oxygen species cause harm to cells (Navari-Izzo et al., 1997). Moreover, as a precursor of PCs, it also serves an important role in plant responses to stress condition caused by heavy metal toxicity (Rauser, 1995; Cobbett and Goldsbrough, 2002). PCs comprise a family of intracellular heavy metal binding peptides with the same basic structure [(γ -L-Glutamyl-LCysteinyl) 2–11- glycine] which are synthesized from GSH by the enzyme phytochelatin synthase (Cobbett et al., 1998; Cobbett, 2000; Nocito et al., 2006).

2.18 Advantages and disadvantages of phytoremediation

Phytoremediation is a remarkable practice compared to other remediation methods, it is not universal and primarily, delivers undamaged biological lively soil. In past, the advantages and restrictions along with its technical aspects have been comprehensively reviewed by various researches (Garbisu et al., 2002; Garbisu and Alkorta, 2003; Alkorta et al., 2004; Epelde, 2010). It is generally considered as an environmentally friendly, efficient, in situ, low-cost, non-invasive, aesthetically agreeable, a socially acknowledged practice, which uses solar driven biological processes to revive heavy metal polluted soils (Alkorta and Garbisu, 2001; Garbisu et al., 2002; Wenzel, 2009). It also recovers the chemical character of the polluted soil by escalating organic matter content, nutrient levels, cation exchange capacity, helps to prevent landscape destruction and enhances activity and diversity of soil microorganisms in order to maintain healthy and self sustaining ecosystems, prevents water and wind erosion as well as runoff and leaching of metals by rhizosphere induced adsorption and precipitation processes (Cunningham and Ow, 1996; Garbisu et al., 2002). Regardless of these advantages, phytoremediation has numerous restrictions that have need of additional rigorous investigation on plants and their ambient soil surroundings.

Phytoremediation has been considered as a continuing approach, it generally takes decades to decrease metal contents present in soil to a secure and satisfactory level representing an important limit for its diffusion (Cunningham et al., 1995; Farid et al., 2014). Another worry connected with the relevance of phytotechnology is its management and removal of contaminated plant waste in an ecofriendly way. Usually, a high cost is a potential drawback to the technology because at first it requires an appropriate collection of contaminated biomass from the field and then subsequent disposal of this hazardous waste in a suitable manner. However, yield of products with economic worth from the selected plant species that has used in the remediation of contaminated soils would be an added profit to phytoremediation, which could certainly facilitate the continuous long term use of this technology (De Paolis et al., 2011). The ambient ecological conditions also decide the competence of phytoremediation as the continued existence and development of plants are unfavorably exaggerated by extreme ecological conditions, toxicity and the broad conditions of soil in polluted fields (Conesa et al., 2011).

Nevertheless, phytoremediation is a lucrative option to the usual old methods (soil excavation, land filling, soil washing). Unlike very costly traditional engineering techniques, such as, stripping the contaminants from the soil using physical, chemical or thermal processes, the total cost of phytoremediation is still lower i.e. comparatively cost effective (Rajakaruna et al., 2006).

3. Materials and Methods

3.1 Material and their sources

Abbildung in dieser Leseprobe nicht enthalten

3.2 Plant material, seed germination and growth conditions

The seeds of Brassica juncea (L.) Czern., Brassica napus L. and Brassica rapa L. were procured from the Krishi Vigyan Kendra, Banasthali University, Banasthali, Rajasthan, India. These seeds were stored in an airtight desiccator containing fused CaCl2 for further use. Before use seeds were surface sterilized with 5 % sodium hypochlorite for 15 minutes and washed thoroughly 4-5 times with distilled water. Seeds were germinated in glass petridishes (10 cm) containing two sheets of autoclaved blotting paper moistened with 10 ml of distilled water at 30 ºC in the dark. Each petridish contained 30 seeds. The cover of petridishes was removed on day 3rd and germinated seeds were transferred to light in thermostatistically controlled culture room maintained at 25 ± 2 ºC and 50 % relative humidity. The sterile water of the petridishes was replaced with Hoagland’s nutrient solution having composition 16 mM KNO3, 4 mM Ca(NO3)2.4H2O, 2 mM NH4H2PO4, 1 mM MgSO4.7H2O, 50 µM KCl, 25 µM H3BO3, 2 µM MnSO4.H2O, 2µM ZnSO4.7H2O, 0.5 µM CuSO4.5H2O, 0.5 µM H2MoO4, 30µM NaFeEDTA. The pH of the Hoagland’s nutrient solution was adjusted to 6.4. Seedlings were provided with photosynthetic photon flux density (PPFD) at 500 µmol m-2 s-1 by a combination of fluorescent tubes and tungsten lamps for 14/10 hours day/night daily.

After 3 days growth in petridishes under above-mentioned conditions, seedlings of uniform size were transferred to hydroponic culture in plastic containers (10×10 cm). Each pot was contained 10 plants in 800 ml of Hoagland’s nutrient solution. The culture conditions were same as described above. The nutrient solution was bubbled with glass rod twice a day to provide sufficient oxygen and mixing of nutrients and was changed on every 3rd day to avoid any nutrient deficiencies to seedlings.

3.2.1 Metal treatment and experimental designs

Subsequent at day 15 of acclimation when seedlings were attained 1-2 leaf stage, they were subjected to different Cu treatments for different experimental setup.15 days old seedlings were treated with for 2 3, 5, 7, and 10 days with following concentrations.

Brassica juncea (L.) Czern., B. rapa L. and Brassica napus L. seedlings- CuCl2 (5, 20, 40, 50, 75 and 100 μM).

After 2 3, 5, 7, and 10 days of treatment the root, leaf of every seedling was separated. A control with Hoagland’s nutrient solution (0 µM) was used.

3.3 Intracellular Cu content determination

Intracellular copper accumulation was determined by the method of Bates et al. (1982). Plants were harvested and washed thoroughly with 20 ml of 2 mM EDTA solution and roots were separated. After oven drying at 80 ºC for overnight dry weight was determined and 100 mg of dried plant material was digested in 5 ml of digestion mixture containing HNO3 (70 %) + H2O2 (30 %) + deionized water in 1:1:3 ratio until the solution become colorless. Residues were dissolved in 2 % (v/v) nitric acid to a final volume of 5 ml and Cu concentration was determined by atomic absorption spectrophotometer (Make: Varian, Model No.: 240 FS). The calculation of intracellular Cu was done by weight by weight and results are reported in mg g-1 of sample. Merck Cu standard was used for quantitation of intracellular Cu content.

The operational parameters of the instrument were set as below.

Abbildung in dieser Leseprobe nicht enthalten

3.4 Plant growth parameters

3.4.1 Determination of germination percentage

To see the effect of Cu toxicity on seed germination was assessed in terms of percentage. After surface sterilization seeds of Brassica juncea, B. rapa and B. napus were put in individual petridishes containing blotting papers soaked in 10 ml of metal solution. Each petridish was contained 30 seeds. Seed germination was observed for all individual concentrations of CuCl2. A control was set without any metal. Triplicate of each concentration were taken. Germination phenomenon was observed for 72 hours. The breaking of the testa was considered as the criterion for germination.

Germination rate (%) = Abbildung in dieser Leseprobe nicht enthalten

3.4.2 Fresh weight, root length and dry weight

Seedlings were harvested and quickly rinsed with deionized water. Metal toxicity and growth was determined by measuring root elongation and plant biomass production (fresh weight and dry weight). Root length was measured and seedlings were marked before the Cu was added to the nutrient solution. After 72 hours of metal exposure, the root length of the same seedlings was again measured and the root growth was expressed as percent decrease of control.

3.4.3 Root elongation rate

The elongation rate was calculated over a period of 3 days in metal Brassica juncea, B. rapa and B. napus seedlings using the technique described by De Koe et al. (1992) and Schat and Bookum (1992). Before starting the metal treatments the longest root was measured. The root system was then marked and being put back into the nutrient solution. After 3 days incubation in CuCl2 containing solution the root length (RL) was again measured. The elongation was obtained by measuring the growth of the longest root. The average elongation rate was calculated from the differences in growth between the beginning and the end of the metal treatment of the longest root. The root elongation rate was calculated ( Parker et al., 1995) as follows:

Elongation rate (cm day-1) = Mean final longest RL-mean initial longest RL

Time of metal exposure

3.4.4 Fresh weight

Fresh weight of the Brassica seedling was recorded before metal treatment and marked individually. Fresh weight of the same samples was again recorded after 72 hours of treatment immediately after the harvesting. Seedling growth was calculated as follows:

Growth rate (FW g day-1) = Abbildung in dieser Leseprobe nicht enthalten

W1: Fresh weight recorded before metal treatment; W2: Fresh weight recorded after metal treatment; T1: Duration before treatment; T2: Duration after treatment

After measuring fresh weight the dry weight (DW) of the same seedlings was recorded. Both were dried in hot air oven at 80 °Cfor overnight. Dry weight was expressed as percent decrease of control.

3.4.5 Tolerance index

A tolerance index (Wilkins, 1978; Baker et al., 1994) was calculated for the fresh biomass as follows:

Tolerance index (%) = Fresh weight of metal treated seedling X 100

Fresh weight of control seedling

All the above observations are the mean ± SE of 3 values (n=3). Measurements were performed on 3 plants per concentration per replicate and the experiment was repeated thrice.

3.5 Estimation of total protein

The protein content of the Brassica juncea (L.) Czern. , Brassica rapa (L.) Brassica napus (L.) seedlings were determined at different Cu concentrations after 2 3, 5, 7, and 10 days of exposure period following the method of Lowry et al. (1951). Bovine serum albumin (BSA) was used as the standard. 0.5 ml of 1N NaOH was followed by 0.1 ml of extracted sample. After 10 minutes digestion on boiling water bath, 2.5 ml of reagent B (48 ml of 5 % Na2CO3 and 2 ml of 0.5 % CuSO4.5H2O in 1 % sodium potassium tartarate) was added. 0.5 ml Folin-phenol reagent was added after 10 minutes. After 30 minutes of incubation a blue colour complex was developed in the mixture. Absorbance was taken at 700 nm against a blank without sample. Protein content was expressed as percent of control.

3.6 Estimation of chlorophyll content

For chlorophyll estimation fresh leaves of treated plant seedlings were collected. The chlorophyll estimation was processed for determination of pigments using the method of Arnon (1949). The chlorophyll content was determined at different metal concentrations after 2 3, 5, 7, and 10 days of treatment. 100 mg of leaves and callus were ground in cold conditions using 80% chilled acetone in dark. The homogenate was centrifuged at 10,000 g for 10 minutes at 4ºC. Absorbance of supernatant was taken at 645 and 663 nm and Total Chl was calculated by using the formula given by Arnon (1949) as follows:

Total chlorophyll (mg g-1 FW) = Abbildung in dieser Leseprobe nicht enthalten

3.7 Extraction and assay of antioxidative enzymes

For extraction 0.5 gm plant sample was homogenized in 5.0 ml extraction buffer containing 1 mM EDTA, 0.05% Triton-X-100, 2% PVP, 1 mM ascorbate in 50 mM phosphate buffer, pH-7.8. This mixture was centrifuged at 13000 rpm for 20 minutes at 4 °C. Resulting supernatant was stored at -20 °C for the assay of different antioxidative enzymes.

3.7.1 Superoxide dismutase assay (EC 1.15.1.1)

SOD activity was assayed by measuring its ability to inhibit the photochemical reduction of nitrobluetetrazolium according to the method of Beauchamp and Fridovich (1971). 3 ml reaction mixture was contained 50 mM phosphate buffer (pH-7.8), 13 mM Methionine, 75 µM NBT, 2 mM Riboflavin, 0.1 mM EDTA and a suitable aliquot of enzyme extract. This reaction mixture was incubated for 30 minutes under fluorescent lamp. A tube containing enzyme kept in dark served as blank while the control tube without enzyme kept in light served as control. The absorbance was taken at 560 nm. One unit of activity is the amount of enzyme required to inhibit 50% initial reduction of NBT under light.

3.7.2. Ascorbate peroxidase assay (EC 1.11.1.11)

Activity of APX is the rate of H2O2-dependent oxidation of ascorbic acid. Its activity was determined in a reaction mixture that contained 50 mM phosphate buffer (pH-7.0), 0.6 mM ascorbic acid and enzyme extract (Chen and Asada, 1989). The reaction was initiated by the addition of 10 µl of 10% H2O2 and the oxidation rate of ascorbic acid was estimated by following the decrease in absorbance at 290 nm for 3min (extinction coefficient 2.8 mM-1 cm-1). The enzyme activity was expressed as unit mg -1 of protein.

3.7.3 Catalase assay (EC 1.11.1.6)

The CAT activity was measured by the method of Aebi (1974). The assay system comprised of 50 mM phosphate buffer (pH-7.0), 20 mM H2O2 and a suitable aliquot of enzyme in the final volume of 3 ml. Decrease in the absorbance was taken at 240 nm. The molar extinction coefficient of H2O2 at 240 nm was taken as 0.039 mM-1 cm-1. The enzyme activity was expressed as unit mg-1 of proteins

3.8 Estimation of hydrogen peroxide (H2O2)

H2O2 production in plant roots treated with Cu for 2 3, 5, 7, and 10 days was determined spectrophotometrically as described by Jana and Choudhuri (1981). H2O2 was extracted by homogenizing plant material in 3 ml of 50 mM phosphate buffer (pH 6.8). Homogenate was centrifuged at 6000 ×g for 25 minutes. To determine H2O2 levels, 3 ml of the above extracted supernatant was mixed with 1 ml of 0.1 % titanium chloride (in 20 % [v/v] H2SO4) and the reaction mixture was centrifuged at 6000 ×g for 15 minutes. The intensity of yellow colour developed was measured at 410 nm. H2O2 was calculated by using the extinction coefficient of 0.28 µmol-1 cm-1.

3.9 Estimation of lipid peroxidation

Lipid peroxidation was determined in Cu treated seedlings for 2 3, 5, 7, and 10 days by the estimation of malonaldehyde (MDA) with 2-thiobarbituric acid (TBA) according to De Vos et al. (1989). Tissues were extracted in 10 ml of 0.25 % [w/v] TBA made in 10 % [w/v] trichloroacetic acid. The mixture was incubated at 95 °C for 30 minutes and then cooled quickly on ice bath. The resulting content was centrifuged at 10,000 ×g for 15 minutes. The absorbance of the supernatant was recorded at 532 and 600 nm. The non- specific absorbance at 600 nm was subtracted from the absorbance at 532 nm. The concentration of MDA content was calculated by using the extinction coefficient of 155 mM-1 cm-1.

3.10 Determination of Ionically bound cell wall peroxidases (CWP)

3.10.1 Cell wall fraction preparation

Cell wall fraction was prepared by homogenizing 30mg tissue in ice cold Sodium acetate buffer (10 mM, pH- 6.0), using 100µl of buffer per mg of original fresh weight of tissue. Homogenate was mixed and centrifuged at 3000g for 15 min at 3ºC. The supernatant was collected and used for assay of soluble peroxidase. Pellet was re-suspended in the same buffer and again centrifuged. This washing procedure was repeated up to six times to ensure that all the soluble peroxidase had been washed out. Supernatant from final wash was assayed for peroxidase activity to confirm their reduction to a negligible level. Finally washed pellet was used as cell wall fraction to extract ionically bound cell wall peroxidase.

3.10.2 Peroxidase extraction

From cell walls, ionically bound cell wall peroxidase was extracted with 1M NaCl (sodium chloride). The washed pellet was re-suspended in 100µl mg-1 original fresh weight of 100mM sodium acetate buffer, pH-6.0, containing 1M NaCl. The suspension was mixed thoroughly and incubated on ice bath for 60 min with periodic shaking. After incubation this sample was centrifuged at 3ºC for 15 min at 3000g. The supernatant contains the salt extractable cell wall peroxidase; this fraction was considered to represent that fraction of peroxidase that is ionically bound to the cell wall in vivo (Goldberg et al., 1987; Bacon et al., 1997).

3.10.3 Peroxidase assay

Cell wall peroxidase was assayed using substrate 3, 3’, 5, 5’-tetramethyl benzidine (TMB) (Bos et al., 1981). TMB was made up at 20 mg ml-1 in DMSO (Dimethylsulphoxide), and stored in aliquots at -20ºC. 5µl of sample was added to each assay tube followed by 100µl of 100mM sodium acetate buffer, pH-6.0 containing 0.1 mg ml-1 TMB and 0.5µl ml-1 of 6% (w/v) H2O2 (Hydrogen peroxide). Mixture was incubated for 60 min and reaction was stopped by 100µl of 0.6M Sulphuric acid (H2SO4) and absorbance was recorded at 450nm. There was no reaction in the absence of hydrogen peroxide. Enzyme activity was calculated by using extinction coefficient of a terminal oxidation product, a yellow diimine that absorb light at 450 nm (Extinction coefficient= 5.9×104 M-1 cm-1).

3.11 Statistical analysis

For graphical representation of the data, Sigma plot 12.5 was used. Statistical analysis of the data was done by using t-test. All the experiments were conducted in triplicates. The data for each treatment were statistically analyzed separately, by analysis of variance (ANOVA) taking P ≤0.05 as significance level.

4A Results (Part-1)

Seedlings of B. juncea (L.) Czern., have been exposed to different concentration of Cu (0, 5, 20,40, 50, 75 and 100 μM) which showed significant reduction in growth rate. Treatment to the seedlings has been given for with a nutrient solution mentioned different concentrations of CuCl2 for 72 hours and changes in biochemical parameters were monitored on day 2,3,5,7, and 10. In present study, level of metal induced stress in metal accumulator plant species Brassica juncea was analyzed.

After exposure to metal, activity of plant defense system has also been explored. In this study, enzymatic and non-enzymatic antioxidants have been assayed along with other important growth parameters.

4A.1 Estimation of growth parameters and intracellular metal Content

After treatment of Brassica juncea (L.) Czern. at different concentrations of CuCl2 for 72 hours, several growth parameters were observed.

In case of root elongation rate of Brassica juncea (L.) Czern., decrease in root elongation rate was noticed as concentration of Cu increase in treatment [Figure A1 and Table 4A.1 (a)]. Significant (P ≤ 0.05) inhibition was observed in 20- 50 μM treated samples in comparison to control sample. The higher inhibition in root elongation rate was observed at 75 μM treated root sample (Table 4A.1).

As table no. 4A.1 (b) mentioned that fresh weight of root sample decrease as Cu concentration increases. A significant decrease (P ≤ 0.05) in fresh weight was observed in 40 and 50 μM treated root samples as compare to control. In case of shoot, fresh weight was also reported in decreasing order as concentration of Cu increase. Significant decrease was reported in 20, 40, 75 and 100 μM treated shoot samples as compare to control. On the other hand, significant decrease was measured in 20- 100 μM treated leaf samples as compare to control. According to table no. 4A.1 (b), a decrease was observed in Translocation index (TI) % of root shoot and leaf sample as concentration of Cu increase. In case of root, TI% was significantly (P ≤ 0.05) decreased in 20- 75 μM treated sample. A significant (P ≤ 0.05) decrease was observed in 20- 100 μM treated sample of shoot. In leaf sample TI % significantly decrease 20- 75 μM treated sample.

Intracellular Cu concentration in B. juncea (L.) Czern after 72 hours in terms of percent of roots, shoot and leaves and their dry weight and percent of total external Cu present in growth medium was observed (Table 4A.2). An increase was observed in dry weight of root, shoot and leaf sample as Cu concentration increase. On the other hand, external Cu% decreased as Cu concentration increase in the treatment.

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