Chapter 1

Review of Literature of Cyanobacteria Metal

Interactions: Physiological and molecular

responses against metal stress

Chapter 1

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1.1 Review of Literature

1.1.1 Cyanobacteria

1.1.1.1. Habitats

Cyanobacteria are widespread in distribution and commonly found in virtually all

ecosystem habitats on Earth, a success that surely reflects their long evolutionary

history. Although algae are primarily aquatic in nature, they are widely and

ubiquitously distributed on our planet, even in some harsh environment that

include hot springs, and deserts, ranging from the hottest to the cold dry valleys of

Antarctica.

Cyanobacteria have been reported to grow in brines, where they form thick mats

at the bottom. Microcoleus chthonoplastes and Oscillatoria species together with

other cyanobacterial components as minor partners, such as Aphanocapsa marina,

Lyngbya aestuarii and Spirulina subsalsa are found in these mats. Thajuddin and

Subramanian (2002) have reported as many as 89 species from the east coast and

69 species from the west coast, of which 56 species were common in both the

habitats. Of a total of 36 species in 16 genera recovered from salt pans of

Pudukkottai District, Tamil Nadu, 18 species exhibited varying degree of salinity

tolerance (45–90 ppt) (Thajuddin and Subramanian, 2002)

Cyanobacteria have been reported from thermal waters all over the world.

Setchell (1903) suggested the upper limit of cyanobacteria as 65 °C to 68 °C and

Lemmermann (1970) described it as 69 °C. Mastigocoleus laminosus,

Phormidium tenue and Synechococcus elongates var. amphigranulatus are the

more common species in hot springs. Cyanobacteria can also tolerate low

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temperatures; Phormidium sp. has been reported from extensive ice layers in the

Antarctic lakes. Taylor (1954) reported Calothrix and Rivularia as common

cyanobacteria inhabiting marine Arctic areas while Gloeocapsa and Nostoc were

abundant in freshwaters. Endolithic cyanobacterial communities are able to trap

and retain water on rock subsurface microenvironments.

A recent account estimated that 700 taxa of non marine algae are present in

Antarctica. The flora is dominated by species of Anabaena, Aphanocapsa,

Calothrix, Chroococcidiopsis, Gloeocapsa, Lyngbya, Mastigocladus,

Microchaete, Microcoleus, Oscillatoria, Phormidium, Plectonema,

Pseudoanabaena, Nodularia, Nostoc, Schizothrix, Scytonema, Stigonema,

Synechococcus and Tolypothrix, (Broady, 1996). Many extremophiles have

evolved to grow best at extremes of pH. Cyanobacteria are indeed present in acid

lakes (pH 4.1–5) and even found to dominate at low pH (Kwiatkowski and Rolft,

1876). This was later on confirmed by a study, which demonstrated the existence

of filamentous cyanobacteria, Chroococcus turgidus, Limnothrix sp.,

Mastigocladus sp., Oscillatoria sp., Spirulina sp. (pH 2.9) and Synechococcus sp.

(pH 4) in acid lakes in Germany (Steinberg et al., 1998).

Extreme alkaliphiles live in soils laden with soda (natron) or in soda lakes where

the pH can rise to 12, but such organisms grow poorly at neutral pH (Grant and

Tindall, 1986). Very often, soda lakes are monospecific, inhabitated by Spirulina

platensis, which serves as human food (single cell protein) of high nutritional

value. Thermophilic cyanobacterial mat communities occur in geothermal springs

of neutral/alkaline pH and at temperatures of up to ~ 74°C. Mat community

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composition is largely temperature-defined, and mats have been clearly

differentiated on the basis of the cyanobacterial taxa (Castenholz, 1996).

1.1.1.2 Ecology of cyanobacteria

Cyanobacteria have a long evolutionary history and documented fossil records

date back to about 3500 million years ago (Schopf, 2000). However, the earliest

DNA-biomarker evidence suggests that cyanobacteria appeared about 2600

million years ago (Hedges et al., 2001). It is widely accepted that ancient

cyanobacteria evolved oxygenic photosynthesis and played a major role in the

change of the oxygen less atmosphere to an oxygenic one (Schopf, 2000).

Additionally, cyanobacteria are believed to have had a considerable effect on the

formation of oxygen rich gas composition of Earth‘s atmosphere (Dismukes,

2001; Paul, 2008), and today the production of oxygen by cyanobacterial

photosynthesis continues to contribute to maintaining the balance of our

atmosphere (Sielaff et al., 2006). Their long evolutionary history is considered as

a reason for the successful survival of cyanobacteria in many habitats and their

wide ecological tolerance (Whitton and Potts, 2000). In addition, cyanobacteria

have developed a wide ecological tolerance to temperature, light, salinity,

moisture, alkalinity, and possess many characteristics and adaptations that explain

their worldwide distribution and success. The distribution of cyanobacteria is

expanded widely on the earth in diverse ecosystems of marine, freshwater, and

terrestrial environments. They are most abundant in aquatic habitats as part of the

plankton, some can be found tightly or loosely attached to surfaces of plants,

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rocks and sediments, and some can be found in hot and acidic springs, in salt

lakes, in deserts, ice shelves, and the arctic (Mur et al., 1999; Rastogi and Sinha,

2009). Cyanobacteria are also important in many terrestrial environments and

they can live in soils or rocks and form symbiotic associations with plants, fungi

and animals (Whitton and Potts, 2000; Oren, 2000; Baracaldo et al., 2005;

Thajuddin and Subramanian, 2005).

The immense diversity within this group of microorganisms, apart from the

variability of morphology and range of habitats, is also reflected in the extent of

their synthesis of natural products. Cyanobacteria have evolved to produce a

diverse array of secondary metabolites that have aided species survival in these

varied and highly competitive ecological niches (Kalaitzis et al., 2009).

Cyanobacteria are commonly associated with the toxic blooms encountered in

many eutrophic fresh and brackish waters and are widely known for their

potential to produce a range of neurotoxic, hepatotoxic, and tumor promoting-

secondary metabolites (Codd et al., 1999; Sivonen and Börner, 2008).

Cyanobacteria are unique phyla that grow in competitive niches and, as a result,

are promising sources of bioactive compounds (Clardy and Walsh, 2004; Lin et

al., 2008).

1.1.1.3 Cyanobacterial physiology and morphology

All cyanobacteria are characterized as eubacteria that grow as autotrophs with

CO2 as the carbon source, utilizing an oxygen-producing photosynthetic

mechanism for the generation of ATP and reductant. Their cellular organization,

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known as prokaryotic, is characterized by the lack of membrane-bound organelles

such as a true nucleus, a chloroplast or a mitochondrion, and resembles that found

in bacteria. Hence the genetic material, the photosynthetic apparatus and the

respiratory system are not segregated by means of internal membranes from the

rest of the cell and their special structure and chemical composition of the cell

wall are basically the same as those of gram-negative bacteria (Stanier and

Cohen-Bazire, 1977; Van den Hoek et al., 1995; Sakamoto et al., 1997;

Castenholz, 2001; Kalaitzis et al., 2009)

They have two photosystems (PSII and PSI) and use water as an electron donor

during photosynthesis, leading to the production of oxygen. Several cyanobacteria

can also perform anoxygenic photosynthesis using only photosystem I if electron

donors such as hydrogen sulphide are present (Madigan et al., 2003).

The principal mode of nutrition, oxygen-evolving photosynthesis, however, is

similar to that which operates in all other nucleate or eukaryotic algae and in

green plants. The photosynthetic pigments of cyanobacteria are located in

thylakoids that lie free in the cytoplasm near the cell periphery. Cell colors vary

from blue-green to violet-red. The green of chlorophyll a is usually masked by

carotenoids (e.g. beta-carotene) and accessory pigments such as phycocyanin,

allophycocyanin and phycoerythrin (phycobiliproteins). The pigments are

embodied in phycobilisomes, which are found in rows on the outer surface of the

thylakoids (Douglas, 1994).

Cyanobacteria show considerable morphological diversity (Whitton and Potts,

2000). They may either be unicellular, be aggregated into flat, spherical, regular

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or irregular colonies, or form single filaments without branches or filaments with

false or true branching. Some cyanobacteria have the ability to produce two types

of specialized cells: (1) heterocysts, which provide the site for nitrogen fixation

and thereby counteract nitrogen demand under conditions of nitrogen deficiency,

and (2) akinetes which are resting cells that allow the species to survive

unfavourable growth conditions. Many species of cyanobacteria possess gas

vesicles, enabling them to regulate their buoyancy and to maintain a certain

vertical position in the water column in response to physical and chemical factors

(Reynolds, 1987; Walsby, 1994). Asexual reproduction of cyanobacteria occurs

by the formation of hormogonia or endospores (exospores are modified

endospores) or by fragmentation of colonies (Lee, 1999).

1.1.1.4 Classification of cyanobacteria

Microbial systematic has long remained an enigma. Conceptual advances in

microbiology during the twentieth century included the realization that a

discontinuity exists between those cellular organisms that are prokaryotic (i.e.

whose cells have no true nucleus) and those that are eukaryotic (i.e. more

complexly structured cells with a nucleus) within the organization of their cells.

The microalgae investigated by phycologists under the International Code of

Botanical Nomenclature (ICBN) (Greuter et al., 2000) included organisms of both

eukaryotic and prokaryotic cell types. The blue green algae constituted the largest

group of the latter category. The prokaryotic nature of these organisms and their

fairly close relationship with eubacteria made work under provisions of the

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International Code of Nomenclature of Bacteria (ICNB) more appropriate

(Rippka et al., 1979; Waterbury, 1992). The most recent taxonomic revision

based on morphological, biochemical and molecular characters is an extensive

compendium by Anagnostidis and Komarek (1985, 1988, 1990). The latest

edition of Bergey‘s Manual (Boone and Castenholz, 2001) places the oxygenic

photosynthetic prokaryotes within the class ‗Cyanobacteria‘, rather than using the

earlier published name Oxyphotobacteria. It classifies the cyanobacteria in ‗form

genera‘. This term, coined by Castenholz (1992), has no standing under the

Bacteriological Code or under the Botanical Code.

Ideally, taxonomy reflects evolutionary relationships of the classified organisms,

and the taxa are monophyletic groups of organisms (Wilmotte and Golubic, 1991;

Wilmotte, 1994). DNA sequences make it possible to infer phylogenies of

organisms (Moritz and Hillis, 1996) and DNA is not affected by environmental

factors in the same manner as many morphological traits are. The 16S rRNA gene

is universally present in bacteria and cyanobacteria and has a conserved function.

Woese and coworkers (Woese et al., 1976; Woese, 1987) established the modern

bacterial phylogenetic classification mainly based on the 16S rRNA gene

sequence. The widely used 16S rRNA region has been useful in several

phylogenetic analyses of cyanobacteria (Wilmotte and Golubic, 1991; Ben-Porath

and Zehr, 1994; Nelissen et al., 1996; Fergusson and Saint, 2000; Wilmotte and

Herdman, 2001). Some of the molecular methods have been also used for

taxonomic studies of cyanobacteria including DNA-DNA hybridization

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(Wilmotte et al., 1997), fingerprinting based on PCR with primers from short and

long tandemly repeated repetitive sequences (Rasmussen and Svenning, 1998),

restriction fragment length polymorphism (RFLP) (Mazel et al., 1990; Asayama

et al., 1996; Lehtimäki et al., 2000), DNA amplification methods (AFLP,

ARDRA, REP-PCR, RAPD) (Neilan, 1995; Satish et al., 2001; Lyra et al., 2001),

sequencing of marker genes, e.g. rpoC1(Fergusson and Staint, 2000), nifH (Ben-

Porath and Zehr, 1994), cpcB and cpcA (Manen and Falquet, 2002; Ballot et al.,

2004; Teneva et al., 2005), ITS region sequencing (the internal transcribed spacer

between the 16S rDNA and 23S rDNA) (Gugger et al.,2002; Orcutt et al., 2002),

PC-IGS region sequencing (phycocyanin operon intergenic spacer) (Neilan et al.,

1995; Bolch et al., 1996; Laamanen et al., 2001; Dyble et al., 2002; Rohrlack et

al., 2008).

1.1.1.5 Biomass and its Use

Algae constitute the first link in the aquatic food chain primarily due to their

autotrophic nature. As a group, they range from extremely small picoplankton to

the giant seaweeds (up to 70 meters long). Approximately 50% of the primary

productivity in aquatic bodies comes from algae and phytoplankton. Aquatic

ecosystems contribute almost the same amount of biomass as the terrestrial

systems and are assumed to incorporate 90-100 gigatons of atmospheric CO2

annually. This biomass in turn determines the fish production and climatological

processes associated with marine productivity (Seigenthaler and Sarmiento, 1993)

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Cyanobacteria play an important role in nutrient recycling and maintenance of

organic matter in the soil but there has been more emphasis on the nitrogen fixing

potential of these organisms. The beneficial effects of cyanobacterial biofertilizer

have also been observed on the physico-chemical properties of soil, improvement

of crop yield, mobilization of nutrients and conservation of the carbon status of

soil. These have been mainly attributed to the production of biologically potent

substances and also extra cellular polysaccharides (Philippis and Vincenzini,

1998). The utilization of cyanobacterial biofertilizer in the rice requires that the

strains are tolerant to a variety of agrochemicals routinely used.

The cyanobacteria have both beneficial and detrimental properties when judged

from a human perspective. Their extensive growth can create considerable

nuisance for management of inland waters (water supply, recreation, fishing, etc.)

and they also release substances into the water, which may be unpleasant or toxic

(Juttner, 1987). The properties that make the cyanobacteria generally undesirable

are also the qualifications for possible positive economic use. Blue-greens are the

source of many valuable products and carry promising physiological processes,

including light-induced hydrogen evolution by biophotolysis (Skulberg, 1993).

Extensive research has taken place in the relevant fields of biotechnology.

1.1.1.6 Sources of oxidative stress in cyanobacteria

1.1.1.6.1 Endogenous Source

In all aerobically living organisms, respiration is thought to be a major source of

ROS produced inside the cells. Molecular oxygen diffuses passively into the cell

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and is reduced to super oxide anion and H2O2 via the oxidation of flavoproteins,

such as the NADH dehydrogenase II (NdhII) in Escherichia coli (Giorgio et al.,

2007). When oxygen comes into contact with metabolic systems it can be

transformed into more reactive forms such as super oxide (O2•), hydrogen

peroxide (H2O2), hydroxyl radical (OH•) and singlet oxygen (1O2) (Smirnoff,

1993). These reactive forms of oxygen (ROS) are continuously produced in living

cells as a by-product of metabolism. In addition to ROS produced by the

respiratory machinery, photosynthetic organisms are challenged by ROS

generated by the photosynthetic electron transport chain. Light is essential for

photosynthesis, but at the same time can also be a source of major stress.

Chloroplasts are considered potentially the most efficient source of ROS. Singlet

oxygen (1O2) are produced in them by oxygen reduction at PS1 and PSII

(Smirnoff, 1993). It is thought to inhibit the repair of PSII inactivated by light.

The production of 1O2 and its impact on photoinhibition increases when the redox

potential of the QA quinine decreases (Fufezan et al., 2007). If light intensity is

higher than that normally managed by the capacity of the photosynthetic electron

flow, not only does 1O2 production increase but other ROS can be formed, leading

to the inactivation of the photosystems. This is because in these cases, oxygen

rather than ferredoxin can be used as an electron acceptor, which generates a

super oxide anion as a primary product. This reaction on the donor side of the PSI

was called the Mehler reaction as it was first described in chloroplasts by Mehler

(1951). Since this pioneering work, it has been assumed that the reduction of

oxygen by the PSI electron flow occurs in all oxygenic phototrophs. However, to

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what extent the Mehler reaction takes place in cyanobacteria is somewhat

controversial. Compared to algae and higher plants, cyanobacteria undergo a high

degree of O2 reduction by consuming 50% of the photosynthetic electrons instead

of only 15% for plants (Badger et al., 2000).

1.1.1.6.2 Exogenous Sources

The rapid increase in urbanization and industrialization is exerting negative

pressure on all kinds of ecosystems. Of these the agroecosystem is more severely

affected by anthropogenic activities such as faulty irrigation practices and

application of pesticides and chemical fertilizers, which in turn increases the

salinity and metal content thereby posing a threat to soil microflora including

cyanobacteria. Moreover, a constant release of gases (CO2, CH4, NOx and CFC)

from various sources leads to enhanced atmospheric temperature. The pollutants

in the atmosphere also lead to depletion of stratospheric ozone as a result of which

there is an increase in UV-B radiations giving rise to another problem of global

concern (Crutzen 1992; Smith 1992). The above-mentioned stresses (salinity,

metals, temperature and UV-B) damage the cells at physiological, biochemical,

and molecular levels (Zhu, 2001).

Cyanobacteria being omnipresent are subject to onslaught of an increased salinity,

metal, high temperature and UV-B radiation. Since cyanobacteria are believed to

be the most primitive photosynthetic microorganism on earth, they have

enormous capacity to adopt against a wide array of stressful conditions like high

salt, high light, UV rays, temperature, metals and pesticides (Whitton and Potts,

2002).

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The understanding of the response of cyanobacteria which can be used as a simple

model for plants to salt stress is of utmost relevance, as deciphering their adaptive

mechanisms will surely contribute not only to understand the plant response but

also to design and construct plants resistant to such an environmental limitation.

Former studies with two strains of Anabaena revealed that salinity-induced

modification of protein synthesis occurs in cyanobacteria, like in plants, and that

some proteins synthesized during salt stress may be essential for cyanobacterial

osmotic adaptation also. Similarly Cyanobacteria also respond to different

temperature ranges and heat exposure. Heat-exposed cyanobacteria exhibit

differential increased expression of two heat shock proteins encoded by the

groEL1 and groEL2 genes. Being phototrophic in nature light also plays a major

role in growth and high light intensity can cause structural and functional

alterations of cyanobacterial phycobilisomes (Tamary et al., 2012).

Presently the response of cyanobacteria towards metal stress is gaining

importance and the results of studies have led these organisms to become

excellent tools for bioremediation. As these oxygen-evolving organisms quickly

respond and adapt to stress conditions in general and heavy metals in particular

there is a possibility to use cyanobacterial dry biomass for remediation of sewage

waters from cadmium (Rangsayatorn, 2002). The results of the study by

Murugesan et al. (2008) also indicated the potentiality of the algae, Spirulina

platensis to be a possible agent for removal of heavy metals from aqueous

solutions.

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1.1.1.7 Metal requirements by the cyanobacteria

Metals are ubiquitous in the biosphere where they occur as part of the natural

constituents of chemicals to which biota and human beings are frequently

exposed. Metals play vital role in mediating important biochemical processes. Fe,

Mg, Cu and Mn are important as micronutrients and are essential cofactors for the

operation of the oxygenic photosynthetic electron transfer apparatus. Zinc (Zn),

Nickel (Ni), Copper (Cu), Vanadium (V), Cobalt (Co), Tungsten (W), and

Chromium(Cr) are toxic elements with high or low importance as trace elements.

Aluminium (Al), Arsenic (As), Mercury (Hg), Silver (Ag), Cadmium (Cd), Lead

(Pb), and Uranium (U) have unknown function as nutrients and seem to be more

or less toxic to plants, algae and micro-organisms (Godbold and Huttermann,

1985; Nies, 2003). The overall metal quota required by photosynthetic organisms

because of oxygenic photosynthetic electron transfer processes is much larger

than the requirement of non-photosynthetic organisms. Oxygenic photosynthesis

exerts unique stresses on photosynthetic organisms. The photosynthetic apparatus

is composed of a number of membrane-embedded protein super complexes that

contain many cofactors.

Among them are Fe cofactors such as iron sulfur (Fe-S) clusters, cytochromes,

and non heme Fe, the Mn cluster of PSII, Cu in plastocyanin, as well as Mg in the

center of each chlorophyll (chl) ring. As a result, the demand for these metals far

exceeds that of the cellular Mn quota of the oxygenic photosynthetic

cyanobacterium Synechocystis sp. PCC 6803 is two orders of magnitude higher

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than that of the nonoxygenic purple bacterium Rhodobacter capsulatus (Keren et

al., 2002). The Fe quota of Synechocystis cells is 1 order of magnitude higher than

that of the non photosynthetic bacterium Escherichia coli (Keren et al., 2004).

Furthermore, more than 25 % of the Fe quota in Synechocystis cells is in PSI

alone (Keren et al., 2004). In plant leaves, 60 % to 80 % of cellular Fe content is

in chloroplasts (Terry and Low, 1982). It is important to note that metal

homeostasis processes are dynamic in nature and the direction of transport

changes in response to availability, annual cycles, and growth phase of

photosynthetic organisms. In the surface water of marine environments, the

concentrations of Fe, Cu, and Mn are often limiting (Morel and Price, 2003). On

land, Fe limits plant growth in arid and semiarid regions (Guerinot, 2001).

Photosynthetic organisms have developed strategies that compensate for metal

limitation. Under Cu limitation, several algae and cyanobacteria species induce

the production of cytochrome c6, which replaces plastocyanin in the

photosynthetic electron transfer pathway (Eriksson et al., 2004). In vascular plant

chloroplasts, where cytochrome c6 is absent, Cu limitation results in a decrease in

copper-zinc (Cu-Zn) superoxide dismutase (SOD) levels (Seigneurin-Berny et al.,

2005). Fe-limited cyanobacteria produce a photosynthetic antenna complex that

facilitates excitation transfer to PSI (Kouril et al., 2005), in effect compensating

for limiting Fe with abundant Mg.

To ensure an adequate supply of metals, photosynthetic organisms from

cyanobacteria to vascular plants have developed efficient strategies for metal

uptake and accumulation (Shcolnick and Keren, 2006). Cavet et al., (2003)

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established that cyanobacteria have metal requirements often absent in other

bacteria; copper in thylakoidal plastocyanin, zinc in carboxysomal carbonic

anhydrase, cobalt in cobalamin, magnesium in chlorophyll, molybdenum in

heterocystous nitrogenase, and manganese in thylakoidal water-splitting oxygen

evolving complex. However, the photosynthetic apparatus presents unique

challenges to metal homeostasis. Whereas metals play a key role as cofactors in

oxygenic photosynthesis, they pose at the same time a major oxidative risk factor

due to their deleterious interaction with oxygen. The extreme redox chemistry

performed by the two photosystems provides multiple sites at which reactive

oxygen species (ROS) can be generated. Cyanobacteria growing in metal-polluted

environments display the ability to tolerate high concentration of toxic metals like

Cu, Cd, and Zn (Mallick et al., 1990)

Therefore, metal transport and storage need to be tightly regulated to ensure

adequate supply and to protect against oxidative damage. The proliferation of all

photosynthetic organisms depends on this delicate balance between the metal

requirements and oxidative damage.

1.1.2 Heavy metals contamination

The ever-increasing population is posing a negative pressure on all kinds of

ecosystems. Particularly the agro ecosystems have been contaminated because of

the application of pesticides and chemical fertilizers resulted in the accumulation

of metals in the soil as well as in the plat tissues (Srivastava et al., 2006).

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The inputs of metals to the environment from anthropogenic activities is

complicated to distinguish as there are very large natural inputs from the erosion,

wind-blown dust, volcanic activity and forest fires. Atmospheric and river inputs,

dredging spoil, direct discharges, industrial dumping and sewage sludge are some

of the important contributors to metal pollution, which lead to the release of metals

to the marine environment.

1.1.2.1 Contamination of aquatic ecosystems

Contamination of aquatic ecosystems by heavy metal is also a worldwide

phenomenon and their levels vary widely depending upon natural and

anthropogenic perturbations (Isani et al., 2009). Our natural waters, particularly

estuaries and fresh water systems are contaminated by fairly long-term pollution

due to metals deposited in sediments from the past (Forstener and wittman, 1981;

Stolzenberg and Drager, 1988) as well as the present human activities. Some

metals enter the sea from the atmosphere, e.g. natural inputs of metals, such as Al

in wind-blowing dust of rocks and shales, and Hg from volcanic activity. As a

consequence of acidification, elevated concentrations of solubilized Al in both

soils and waters have become a serious problem in industrial areas. Pb inputs in

the atmosphere from industrial and vehicular exhaust are much greater than

natural inputs. Some metals are deposited by gas exchange at the sea surface, by

fallout of particles (dry deposition) or are scavenged from the air column by

precipitation (rain), which is called wet deposition. Rivers make a major

contribution of metals in the marine environment (Schindler, 1991).

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Effluents contain metal from various electroplating, tanning, pesticide and nuclear

reactor, which are present as ionic species, inorganic or organic complexes or

associated with colloides and suspended particulate materials. Being non-

degradable, they persist in the environment and accumulate in different parts of

living organisms (Jain et al., 1998).

The Cd has been recognized as one of the most toxic aquatic contaminants and its

concentration ranges from 0.1 mg L-1

in open ocean water to several mgL-1

in

coastal areas with industrial establishments (Husaini and Rai, 1991). Very high

concentrations (5 to 120 µg mL-1

) of this metal are reported from Indian aquatic

(Mathur et al., 1987). Toxic levels of Cu have been reported in the aquatics due to

the anthropogenic activities, such as mining, smelting, waste disposal, use of Cu

containing algicide, metal containing fertilizers, organic manures, and fungicides

(Yruela, 2005; Bona et al., 2007)

1.1.2.2 Contamination of terrestrial ecosystems

In most terrestrial ecosystems, there are two main sources of heavy metals: the

underlying parent materials and the atmosphere. The concentrations of heavy

metals in soils depend on the weathering of the bedrock and on atmospheric

inputs of metals. Natural sources are volcanoes and continental dusts.

Anthropogenic activities like mining, combustion of fossil fuels, metal-working

industries, phosphate fertilizers, etc., lead to the emission of metals and the

accumulation of these compounds in ecosystems (Lantsy and Mackensie, 1979;

Galloway et al., 1982). Thus Agricultural soils in many parts of the world are

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slightly to moderately contaminated by metals such as Cd, Cu, Zn, Ni, Co, Cr, Pb

and As. It has been estimated that, for example, the anthropogenic emissions of

Cd are in the range of 30 000 tonnes per year (di Toppi and Gabbrielli, 1999). Al

is the most abundant metal and the third most abundant element in the earth‘s

crust after oxygen and silicon, comprising approximately 7% of its mass (Foy et

al., 1978). At mildly acidic or neutral soils, it occurs primarily as insoluble

deposits and is essentially biologically inactive (Kochian et al., 2004). However,

Al toxicity is one of the most deleterious factors for plant growth in acidic soils,

which comprise up to 70% of the world‘s potentially arable lands (Von Uexkull

and Mutert, 1995). Furthermore, the acidity of the soils is gradually increasing as

a result of the environmental problems including some farming practices and acid

rain.

Cu is a ubiquitous pollutant in the environment due to the emission and

atmospheric deposition of metal dust released by human activities. In addition,

soils may contain elevated levels of copper because of its widespread use as a

pesticide, land application of sewage sludges as well as mining and smelting

activities (Alaoui-Sosse et al., 2003).

Mercury (Hg), one of the non-essential heavy metals for plants, is frequently

reported to be released into the biosphere including air, waters and soils (Patra

and Sharma, 2000). Due to its transition properties, mercury is readily uptake by

plants, accumulates at high level, and consequently results in toxicity or even

death of plants (Boening, 2000; Patra and Sharma, 2000; Esteban et al., 2008).

Numerous studies have shown that Hg-induced toxicity in plants results from the

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binding of its ionic forms (Hg2+

) to sulphydryl groups of proteins, disruption of

structure, and displacement of essential elements (Van Assche and Clijsters, 1990;

Hall, 2002; Schützendübel and Polle, 2002).Thus the accumulation of such

components, which do not constitute a part of any biogeochemical cycle, is

obviously harmful.

1.1.3 Metal Uptake and Interactions

Algae in metal containing localities tend to concentrate metal from ambient water

and pass them to higher trophic level. They form the base of the food chain and

their primary productivity depends upon maintaining the level of available metal

ion at a concentration between toxicity and deficiency. Accumulation of trace

metals in the food chain has been considered as a major environmental hazard

(Rao and Govindarajan, 1992). Being important in primary production the study

of toxic effects of metal pollutant on algae is important.

Uptake systems for essential metal ions have to differentiate between ions that are

structurally very similar. Therefore most cells have two types of import systems:

those that are nonspecific and are able to import more than one species across the

cytoplasmic membrane and those with high substrate specificity. The first are

faster and driven by the chemiosmotic gradient across the cytoplasmic membrane.

The second often uses ATP hydrolysis as the energy source and its transporters

usually are ABC-type or P-type ATPases (Nies, 2003). A ubiquitous super family

of carriers capable of transporting small solutes in response to chemiosmotic ion

gradients is the major facilitator super family (MFS), whose elements are

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constituted by single-polypeptides transporters (as opposed to the energy-

requiring pumps, that usually are multicomponent transporters) (Pao et al. 1998).

The presence of these transporters has been established in Synechocystis sp. PCC

6803, and their occurrence is thought to be widespread through the cyanobacterial

group (Paulsen et al., 1998). The ABC transporters translocate a variety of

biological molecules across cell membranes, such as peptides and amino acids,

sugars and ions in general (Mikkat and Hagemann, 2000; Holland and Blight,

1999). Microbial genome analysis has shown putative ABC transporters in

Synechocystis sp. PCC 6803 as one of the most conspicuous super families of

membrane carriers (Paulsen et al., 1998). Concretely it has been shown that, in

this species, a high affinity ABC-type Mn transport system is involved in

regulating the uptake of this element (Rukhman et al., 2005). Based on genome

sequence information and similarity to Escherichia coli genes, an ABC-type Mo

transporter is assumed to exist in Synechocystis sp. (Self et al., 2001). An ABC-

type transporter specific for Zn is thought to occur in Synechocystis sp. PCC 6803

(Cavet et al., 2003). A P-type ATPase is a ubiquitous membrane transporter that

carries metal ions, being a mechanism for the control of cytoplasmic metals

(Arnesano et al., 2002). Their distinguishing feature is the formation of a

phosphorylated intermediate during the reaction cycle (hence P-type) (Axelsen

and Palmgren, 1998). The division of this super family into five major branches:

Type I ATPases (heavy metal pumps), Type II ATPases (Ca—ATPases, Na/K—

ATPases, and H/K—ATPases), Type III ATPases (H and Mg pumps), Type IV

ATPases (phospholipids pumps), and Type V ATPases (a group of pumps having

Chapter 1

Page 21

no assigned substrate specificity) and the analysis of conserved core sequences in

several organisms has help to identify putative ATPases in cyanobacteria

(Axelsen and Palmgren, 1998). A Type I ATPase with presumable affinity for K

has been discovered in Synechocystis sp. PCC 6803; another with affinity for

Cu(II) has been discovered both in Synechocystis sp. PCC 6803 and

Synechococcus sp. PCC 7942, the ion specificity of the later being experimentally

shown (Axelsen and Palmgren, 1998). A putative Type II ATPase with affinity

for Ca is present in both mentioned species (Axelsen and Palmgren 1998) and a

putative Na ATPase has been reported for Synechocystis sp. PCC 6803 (Matsuda

et al., 2004; Wang et al., 2002) and Anabaena sp. PCC 7120 (Blanco-Rivero et

al., 2005).

Besides the proteins that transport metals through the membrane there are also

smaller soluble proteins that deliver metals to specific target proteins. Among

these are the soluble metal receptor proteins, known as metallochaperones, that

deliver the metal ion to its proper destination by ensuring that adventitious

reactions and binding to sites other than the appropriate ones do not take place

(O‘Halloran and Cullota, 2000). The lack of intracellular compartments in

prokaryotes accounted for the prediction that no such chaperones would occur in

these organisms. However, they were found in the gram-positive Enterococcus

hirae—a Cu chaperone-like protein (Odermatt and Solioz, 1995) and since then

related proteins have been found in other bacteria groups including the

cyanobacterium Synechocystis sp. PCC 6803 (Banci et al., 2004). Evidence that

Ni metallochaperones assist in the delivery of ions to target proteins, or target

Chapter 1

Page 22

compartments, has been growing (Mulrooney and Hausinger, 2003). For many

elements there is evidence that bacteria control their metal requirements by the

expression of metalloproteins (Borrely et al., 2004). Metal-responsive

transcriptional regulators modulate expression of the genes encoding metal ion

binding and/or transport proteins according to the respective substrate

concentration (Cavet et al., 2002), thus allowing metal ion homeostasis to be

maintained in conditions of either metal ion limitation or excess (Silver, 1998).

Metalloproteins are able to acquire the correct metals due both to selective co-

ordination environments and to the selectivity of metal sensors, metal

transporters, sequestration proteins and metallochaperones but the role played by

each of these factors is still not clear (Cavet et al., 2003).

Utilization of phytoplanktonic algae with a high potential to adsorb heavy metals

for the removal of residual metals from waste water resulting in high quality

reusable efficient water and valuable biomass that could be used for different

purpose. As cyanobacteria also have the capability to accumulate, detoxify, or

metabolize such contaminants, to some extent (Garcia-Meza et al., 2005; Wang et

al., 2005; Feris et al., 2004; Pawlik-Skowronska et al., 1997; Koelmans et al.,

1996) they can be used as efficient tool for studying alterations in various

biochemical and molecular processes in organisms under metal stress.

Cyanobacteria are characterized by high tolerance and can exist in various

extreme conditions: in hot springs, in snow, in salts, etc. Toxic influence of heavy

metals to aquatic organisms depends not only on metal concentration but also on

the chemical form of their occurrence (speciation) (Sunda, 1990); however, the

Chapter 1

Page 23

speciation depends on a number of factors (e.g., pH, temperature, salinity,

concentration of inorganic ions, dissolved organic matter) (Heng et al., 2004;

Zirino and Yamamoto, 1972; Crist et al., 1981).

1.1.3.1 Metal and Oxidative stress

The primary response of organisms is the generation of reactive oxygen species

(ROS) upon exposure to high levels of heavy metals. Various metals either

generate ROS directly through Haber-Weiss reactions or indirectly by interaction

with the antioxidant system (Srivastava et al., 2005), disrupting the electron

transport chain (Qadir et al., 2004) or disturbing the metabolism of essential

elements (Dong et al., 2006).

Fig 1.1 Site of free radical generation in cyanobacteria (Shcolnick and Keren,

2006)

Chapter 1

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ROS are generally very reactive molecules possessing an unpaired electron.

Under standard growth conditions, ROS levels in an organism are under tight

control of scavenging systems that include enzymatic and non-enzymatic

antioxidants however, when ROS are not adequately removed, an effect termed

―oxidative stress‖ may result. Excess ROS formed within cells can provoke

oxidation and modification of cellular amino acids, proteins, membrane lipids and

DNA. These changes lead to oxidative injuries and result in the reduction of plant

growth and development (Ogawa and Iwabuchi, 2001).

Fig 1.2 ROS induced oxidative Damage (Kohen and Nyska, 2002)

When oxygen comes into contact with metabolic systems it can be transformed

into more reactive forms such as super oxide (O2•), hydrogen peroxide (H2O2),

Chapter 1

Page 25

hydroxyl radical (OH•) and singlet oxygen (1O2) (Smirnoff, 1993). These reactive

forms of oxygen (ROS) are continuously produced in living cells as a by-product

of metabolism (Bowler et al., 1992; Scandalios 1993). Transition metals like Cu

and Fe catalyze the formation of OH• in the Fenton and metal-catalyzed Haber-

Weiss reactions (Hall, 2002). These radicals can also be formed from H2O2 and

triplet state of chlorophyll (Ramalho et al. 1998; Alscher et al., 1997). In all

aerobically living organisms, respiration is thought to be a major source of ROS

produced inside the cells. Molecular oxygen diffuses passively into the cell and is

reduced to super oxide anion and H2O2 via the oxidation of flavoproteins, such as

the NADH dehydrogenase II (NdhII) in Escherichia coli (Imlay, 2003; Giorgio et

al., 2007). In addition to ROS produced by the respiratory machinery,

photosynthetic organisms are challenged by ROS generated by the photosynthetic

electron transport chain. Light is essential for photosynthesis, but at the same time

can also be a source of major stress. Chloroplasts are considered potentially the

most efficient source of ROS (Bartosz, 1997). Superoxide radical (O2•) and

Singlet oxygen (1O2) are produced in them by oxygen reduction at PS1 and PSII

(Smirnoff, 1993). 1O2 is produced by an energy input to oxygen from

photosensitized chlorophyll. It is thought to inhibit the repair of PSII inactivated

by light. The production of 1O2

and its impact on photoinhibition increases when

the redox potential of the QA quinine decreases (Fufezan et al., 2007). If light

intensity is higher than that normally managed by the capacity of the

photosynthetic electron flow, not only does 1O2 production increase but other

ROS can be formed, leading to the inactivation of the photosystems. This is

Chapter 1

Page 26

because in these cases, oxygen rather than ferredoxin can be used as an electron

acceptor, which generates a super oxide anion as a primary product. This reaction

on the donor side of the PSI was called the Mehler reaction as it was first

described in chloroplasts by Mehler (1951). Since this pioneering work, it has

been assumed that the reduction of oxygen by the PSI electron flow occurs in all

oxygenic phototrophs. However, to what extent the Mehler reaction takes place in

cyanobacteria is somewhat controversial. Compared to algae and higher plants,

cyanobacteria undergo a high degree of O2 reduction by consuming 50% of the

photosynthetic electrons instead of only 15% for plants (Badger et al., 2000).

ROS accumulation induces oxidative processes such as membrane lipid

peroxidation, protein oxidation, enzyme inhibition and DNA and RNA damage,

resulting in cell damage and, eventually, cell death (Keshari et al., 2011;

Hammond-Kosack and Jones, 1996).

1.1.3.2 Mechanisms of Metal Toxicity in Cyanobacteria

The mechanisms by which metals exert their toxicity in living organisms is very

diverse, especially their involvement in oxidative biochemical reactions through

the formation of ROS (Goyer, 1991). Molecular mechanisms of heavy metal

cytotoxicity include damage to plasma membranes, following binding to proteins

and phospholipids, inhibition of Na, K dependent ATPases, inhibition of

transmembrane amino acid transport, enzyme inhibition, lipid peroxidation and

oxidative DNA damage, depletion of antioxidant enzymes (such as glutathione)

through the generation of ROS (Stohs and Bagchi ,1995; Sigel and Sigel, 1992).

Chapter 1

Page 27

Metal ions can penetrate inside the cell, interrupting cellular metabolism and in

some cases can enter the nucleus. Metal cations can also bind to DNA through

ionic and coordinated bonds in a reversible way, but can not produce all the

lesions observed in chromatin of cells. Hence, not only the direct, but mostly

indirect effects of metals on nuclear chromatin must be considered more

important in DNA damage.

Exposure to elevated concentrations of heavy metals results in growth inhibition

(Marschene, 1995). Furthermore, heavy metal, have been demonstrated to reduce

light harvesting pigments (Tripathi et al., 1981; Xylander and Braune, 1994) as

well as changes in photosynthetic electron transport system (Singh et al., 1991;

Husaini and Rai, 1991). Certain heavy metals, viz. Cd, Ni, Hg and Cr, are to

inhibit growth, pigment synthesis, nutrient uptake, nitrogen fixation and

photosynthesis in Anabaena inaequalis, Anabaena doliolum and Nostoc

muscorum (Stratton et al., 1979; Rai and Raizada, 1985).

Cd toxicity to algae and cyanobacteria has been extensively studied by selecting

such parameters as growth (Conway and Williams, 1979; Sakaguchi et al., 1979;

Stratton and Corke, 1979), carbon fixation (Hart and Scaifes, 1977; Wong et al.,

1979), nitrogenase activity (Stratton and Corke, 1979) and ultra structural changes

(Rachlin et al., 1982; Rai et al., 1990), nutrient uptake and enzyme involved in

their metabolism (Rai et al., 1998). Loss of chlorophyll and carotenoids as a

consequence of heavy metal exposure of eukaryotic green algae as well as

cyanobacteria is well known (Rosko and Rachlin, 1975; Rai et al., 1990, 1991).

The decrease in photosynthetic pigments is due to the lysis of cell wall and

Chapter 1

Page 28

disruption of the thylakoid membrane as reported for Anabaena flos aquae (Rai et

al., 1990). However, photosynthetic pigments were also found to be reduced in

Cu treated culture (Mallick and Rai, 1990).

De Filippis et al., (1981) reported that the reduction in chlorophyll a content is a

common symptom of heavy metal toxicity. Heavy metals like Arsenic As at 100

μg/l or above have been found to affect N2-fixing activity of symbiotic

cyanobacteria Anabaena azollae (Aziz, 2001). He also observed negative effect of

as on chlorophyll a and b synthesis and increased anthocyanin formation in Azolla

filiculoides as the concentration of As is increased.

El-Naggar et al., (1999) reported that lower concentrations of heavy metal (Co2+

)

stimulate growth of Nostoc muscorum, followed by inhibition at higher

concentrations. At low concentrations, substitution of Pb2+

for Zn2+

in some

metalloenzymes in vitro and in vivo may result in growth promotion (El-Sheekh et

al., 2005). Further, Pb is a toxic metal with no biological function (Allen, 1997).

Hemlata and Fatma, (2009) found lead to be more toxic than nickel, copper and

zinc metal to various strains of Anabaena. Toxicological responses of

cyanobacteria to lead exposure range from sensitive to tolerant (Khalil, 1994; Rai

et al., 1996, 1998). Spirulina platensis contains detectable amount of Cu, Zn and

Pb when grown under contaminated condition (Choudhary et al., 2006). Rana et

al., (2010) reported that the growth rate of the isolated species of Lyngbya showed

a gradual decrease at high concentration of lead.

Photosynthetic functions have been invariably affected either directly or indirectly

by heavy metals (Krupa and Baszynski, 1995; Prasad et al., 2002), since

Chapter 1

Page 29

thylakoids are the photosynthetic lamellae of cells containing almost all cellular

chlorophylls and carotenoids. Heavy metals are reported to inhibit electron

transport chain, both the PSI and PSII are inhibited by metals but the site of the

inhibition of heavy metals was closer to the PSII reaction center. Lu et al. (2000)

demonstrated that chlorophyll fluorescence analysis could be a useful

physiological tool to assess early stages of change in photosynthetic performance

of algae in response to heavy metal pollution.

The inhibition may be additional at plastoquionon level as reported for Zn isolated

barley chloroplasts (Tripathi and Mohanty, 1980) or by inhibition of electron

transport at the oxidizing side of PSII as reported for Cr, Ni and Pb (Singh et al.,

1991; Prasad et al., 1991). Babu et al., (2010) reported inhibition of PSII activity

with increasing concentration of Cr and Ag. El-Sheekh et al., 2003 demonstrated

that the inhibitory action of Co is located on the acceptor side of PSII for both M.

minutum and N. perminuta. Besides PSII activity, PSI was also inhibited by

heavy metal (Singh et al., 1991). Wong and Govindjee (1976) reported Pb

induced inhibition of PSI in isolated bundle sheath and mesophyll chloroplasts of

Zea mays, however, Cd inhibited electron flow on the reducing side of PSI.

The effect of Ni toxicity on biomass and cell density of Anabaena doliolum was

studied by Shukla et al. (2009). Ni is a transition metal and found in natural soils

at trace concentrations except in ultramafic or serpentinic soils. However, Ni2+

concentration is increasing in certain areas by human activities such as mining

works, emission of smelters, burning of coal and oil, sewage, phosphate fertilizers

and pesticides (El-Enany and Issa, 2000). Concentration of Ni in polluted soil

Chapter 1

Page 30

may reach 20- to 30-fold (200–26,000 mg/kg) higher than the overall range (10–

1000 mg/kg) found in natural soil (Izosimova, 2005). Excess of Ni2+

in soil causes

various physiological alterations and diverse toxicity symptoms such as chlorosis

and necrosis in different plant species (Pandey and Sharma, 2002; Rahman et al.,

2005), including rice (Samantaray et al., 1997). The Ni also affected the lipid

composition and H-ATPase activity of the plasma membrane as reported in Oryza

sativa shoots (Ros et al., 1992. Exposure of cyanobacteria to high concentration

of metal for a long time decreased the biomass and cell density.

Cu and Zn ions each produce a slight decrease in oxygen evolution whereas the

greatest effect on oxygen evolution was produced by cadmium ions as reported by

Ybarra and Webb (1999). Cu is known to be an essential micronutrient for plants

and algae since the pioneer works of Arnon and Stout (1939). It is an essential

element as it is involved in a number of physiological processes such as the

photosynthetic and respiratory electron transport chains (Van Assche and

Clijsters, 1990) and as a cofactor or as a part of the prosthetic group of many key

enzymes involved in different metabolic pathways, including ATP synthesis

(Harrison et al.,1999). Higher plants take up Cu from the soil solution mainly as

Cu2+

. In excess, the absorbed copper can be considered as a toxic element leading

to growth inhibition (Ouzounidou et al., 1992). Many studies have been carried

out on the effect of Cu excess on growth, mineral nutrition and metabolism of

plants. Cu excess reduces growth (Maksymiec et al., 1995), photosynthetic

activity (Lidon et al., 1993) and the quantum yield of PSII photochemistry

Chapter 1

Page 31

assessed by chlorophyll fluorescence (Maksymiec et al., 1999). Lidon et al., 1999

suggested that the primary sites of copper inhibition are the antenna chlorophyll

molecules of PSII.

Fig 1.3 Scheme of toxic Cu action sites in photosystem II (Yruela et al., 2005)

A. flos-aquae strain was sensitive to increased Cu concentration and inhibited

growth of the tested cyanobacterium. Chlorophyll a concentration decreased with

increased metal concentration Surosz (2004). The effects of Zn and Cd on the

unicellular Cyanophyta Chroococcus minutus indicated that each metal depressed

the growth rate as reported by Battah (2010).

Chapter 1

Page 32

Due to the increasing number of reports on toxic effects of Al on all forms of

biological life, the search for mechanisms of the adverse effects of Al has been

intensified. The only oxidation state is +III and any binding of Al will therefore

mainly be electrostatic. Al3+

associates with several low molecular weight ligands,

preferentially oxygen-donating ligands such as phosphate and carboxylate groups.

Al3+

has an especially strong affinity for phosphate ions as well as for organic

phosphorus compounds (Guo et al., 2010).

Tamas et al., (2005) reported that Al-induced root growth inhibition correlated

with Al uptake and cell death. Horst (1999) supposed that binding of Al to

sensitive binding sites of the apoplast and competition for these binding sites with

other ions determines Al-induced inhibition of root elongation. These rapid Al-

induced changes in cell wall can lead to the inhibition of water and mineral uptake

imitating drought stress. In addition, the rigid structure of the plasma membrane

caused by metal toxicity can also affect the uptake of water and ions (Fodor et al.,

1995). The same mechanism of Al toxicity was also reported by Tamas et al.,

(2006). Their results suggested correlation between Al uptake, Al-induced

drought stress, oxidative stress, cell death and root growth inhibition in barley

roots.

Toxic effects of Al on plants are well known (Kollmeier et al., 2000; Zhou et al.,

2009). Accumulating evidence shows that Al toxicity affects light absorption

(Chen et al., 2005a), photosynthetic electron transport (Chen, 2006; Moustakas et

al., 1996); gas exchange (Chen et al., 2005b; Hoddinott and Richter, 1987;

Pereira et al., 2000; Jiang et al., 2008), photo protective systems (Xiao et al.,

Chapter 1

Page 33

2003a; Chen et al., 2005a; Ali et al., 2008), pigments (Chen et al., 2005a;

Mihailoovic et al., 2008; Milivojevi et al., 2000), ultrastructure (Moustakas et al.,

1996; Konarska, 2010; Peixoto et al., 2002; Xiao et al., 2003b), carbohydrates

(Chen et al., 2005b; Graham, 2002) and photosynthetic enzymes (Chen et al.,

2005b) in plant leaves. Peixoto et al. (2002) reported that, the photosynthetic rate

was affected by Al toxicity to a greater extent in Al-tolerant than in Al-sensitive

sorghum cultivar. As a result of thylakoid degradation, increasing Al

concentration inhibits photosynthesis (Haug and Foy, 1984). Pettersson et al,

1985b, 1986 demonstrated the very rapid accumulation of Al into the

cyanobacterium Anabaena cylindrica which made this organism suitable for

studies of intracellular actions of Al and reported that, both accumulation of

cyanophycin granules and degradation of thylakoids were the most pronounced

ultra structural changes induced by Al in Anabaena cylindrica. Al, at 10 µM,

could result in severe injuries to chloroplast membranes of spinach (Spinacia

oleracea; Hampp and Schnabl, 1975). Konarska (2010) reported that, the

mesophyll cells from Al-stressed red pepper (Capsicum annuum) leaves contained

enlarged chloroplasts having a disturbed lamellar system, filled with large starch

grains and rounded mitochondria characterized by the electron lighter matrix and

the degradation of cristae.

1.1.3.3 Molecular Targets of Oxidative Damage

At high concentration, metals catalyze the synthesis of ROS which not only

causes alterations in the growth, physiology and pigments of cyanobacterial cells

Chapter 1

Page 34

but also damages the most important macromolecules like lipids, proteins and

DNA. (Srivastava et al., 2005)

Fig 1.4 ROS production and targets (Latifi et al., 2008)

1.1.3.3.1 Lipids

One of the most deleterious effects induced by heavy metals exposure in plants,

algae and cyanobacteria is lipid peroxidation, which can directly cause

biomembrane deterioration. Malondialdehyde (MDA), one of the decomposition

products of polyunsaturated fatty acids of membrane is regarded as a reliable

indicator of oxidative stress (Demiral and Turkan, 2005). All cellular membranes

Chapter 1

Page 35

are especially vulnerable to oxidation due to their high concentrations of

unsaturated fatty acid. The damage to lipids, usually called lipid peroxidation,

occurs in 3 stages. The first stage, initiation, involves the attack of a reactive

oxygen metabolite capable of abstracting a hydrogen atom from a methylene

group in the lipid. When oxygen is in sufficient concentration in the surroundings,

the fatty acid radical will react with it to form ROO. during the propagation stage.

These radicals themselves are capable of abstracting another hydrogen atom from

a neighbouring fatty acid molecule, which leads again to the production of fatty

acid radicals that undergo the same reactions—rearrangement and interaction with

oxygen (Kohen and Nyska, 2002).Lipid peroxidation is linked to the production

of O-2. Presence of high amounts of transitional metals such as Cu or Fe would

also favour enhanced generation of OH• from O2• through the Fenton reaction.

Thus, the increased level of MDA suggests that metal ions stimulate free radical

generating capacity of the microorganism. Increased MDA level has been reported

in several higher plants also (Chaoui et al., 1997; Luna et al., 1994).

Changes in thiobarbituric acid reactive substances (TBARS) concentration in a

tissue can be a good indicator of the structural integrity of plant membranes. Its

level increased three fold in oat leaf segments treated with 100 μmol Cu (Luna et

al.,1994). The response was less pronounced but also highly significant in intact

plants, for example about 50% in wheat roots treated with 15 μmol l-1

Ni for 6h

(Pandlfini et al.,1992) and 15 to 50% in roots, stems or leaves of bean plants

exposed to 5 μmol l-1

Cd or 100 μmol l-1

Zn (Chaoui et al., 1997). Posmyk et al.,

(2009) showed that high concentration of Cu2+

(2.5 mM) in the medium induced

Chapter 1

Page 36

TBARS accumulation, which was gradually increasing with the time of exposure.

Lipid peroxidation expressed as MDA level was shown to increase under heavy

metal stress (Gallego et al., 1996; Cho and Park 2000; Shah et al., 2001).

Alterations in Lipid Profile under stress

Prokaryotic membranes contain diverse lipid molecular species, and the lipid

composition changes in response to both internal and external cues. Knowing how

lipid molecular species change and how the changes are generated is important to

the understanding of membrane and cell functions. The unsaturated fatty acids of

cell membrane are primary targets of peroxidation which subsequently leads to

cell death, whereas organisms employ numerous approaches to limit their

damage. Hence, a low degree of fatty acid unsaturation may protect the cell

against oxidative damage. There have reports dealing with such adaptations in

fatty acid content under Cr (VI) induced oxidative stress (Kumar et al., 2011).

Other abiotic stresses like UV rays also induce changes in lipid profile. Bhandari

and Sharma, (2006) observed UV-B induced changes in profile of phospho-

glycolipids and neutral lipids of P. corium.

1.1.3.3.2 Proteins

Proteins, also major constituents of membranes, can serve as possible targets for

attack by ROS (Davis, 1987). Among the various ROS, the OH., alkoxy (RO

.) and

nitrogen-reactive radicals predominantly cause protein damage. Hydrogen

peroxide itself and super oxide radicals in physiological concentrations exert

Chapter 1

Page 37

weak effects on proteins; those containing SH groups, however, can undergo

oxidation following interaction with H2O2. Proteins can undergo direct and

indirect damage following interaction with ROS, including peroxidation, damage

to specific amino-acid residues, and changes in their tertiary structure,

degradation, and fragmentation. The consequences of protein damage as a

response mechanism to stress are loss of enzymatic activity, altered cellular

functions such as energy production, interference with the creation of membrane

potentials, and changes in the type and level of cellular proteins (Duikan et al.,

2000).

Following protein oxidation, modified proteins are susceptible to many changes in

their function. These include chemical fragmentation, inactivation, and increased

proteolytic degradation (Stadtman, 1986). SDS-PAGE analyses of the whole-cell

protein after cell exposures to the various Cu concentration demonstrated gradual

decrease in protein content of almost every band with the increase of the metal

concentration (Cu and Cd) in Anabaena flos-aquae (Surosz and Palinska, 2004).

1.1.3.3.3 DNA

Being prokaryotic in nature the genetic material of cyanobacteria are not

segregated by means of internal membranes from the rest of the cell. The limited

chemical stability of DNA under these conditions is considered to be the major

factor making this molecule vulnerable to the attack of ROS.

Chapter 1

Page 38

ROS can interact with it and cause several types of damage: modification of DNA

bases, single- and double-DNA breaks, loss of purines (apurinic sites), damage to

the deoxyribose sugar, DNA-protein cross-linkage, and damage to the DNA repair

system. Not all ROS can cause damage; most is attributable to hydroxyl radicals.

For example, following exposure of DNA to hydroxyl radicals, like those induced

by ionizing irradiation, a variety of adducts are formed. The OH. can attack

guanine at its C-8 position to yield an oxidation product, 8- hydroxyl

deoxyguanosine (8-OHdG) (Jaffe, 1976). The direct interaction of DNA with

other less reactive ROS, such as O2• and H2O2, does not lead to damage at their

physiological concentrations; however, these species serve as sources for other

reactive intermediates that can easily attack and cause damage. For example,

H2O2 and super oxide might lead to the production of the OH. via the Haber-

Weiss reaction, and NO• and O2• might lead to the formation of ONOO• that can

easily cause DNA damage similar to that obtained when hydroxyl radicals are

involved. Transition metals like iron that possess high-binding affinity to DNA

sites can catalyze the production of OH. in close proximity to the DNA molecule,

thus ensuring repeated attack upon the DNA by an efflux of hydroxyl radicals

(Ames, 2001, Halliwell, 1999; Poulsen et al., 2000; Kasprzak, 2002). Metals like

Al exert its toxicity by an altogether a different mechanism. As discussed in the

Section 1.1.3.1 Al creates dehydration stress and it was proved by Shirkey et al.,

(2003) that free radical DNA damage was not a significant event during

desiccation thus indicating that when dry, the DNA is protected from free radical

damage.

Chapter 1

Page 39

Metal stress induces programmed cell death in aquatic organisms

Amongst the aquatic organisms the molecular PCD pathways in filamentous fungi

are well known. The available data have clearly demonstrated the presence of an

ancestral apoptotic machinery in these organisms (Robson, 2006).

Although UV, high light, salt and dessication stress mediated PCD in

cyanobacteria are available in literature, metal mediated PCD in cyanobacteria are

only now starting to be uncovered. DNA strand breaks are observed widely in

cells under UV-B irradiation (Slieman and Nicholson, 2000; Sato et al., 1996;

Lloyd, 1993; Kleiman et al., 1990; Reddy et al.,1998; Blakefield and

Harris,1994). In the cyanobacterium Anabaena sp., a significantly increased

number of DNA strand breaks under UV-B stress was detected by the FADU

method. (Baumstark-Khan et al., 2000.) The exogenous addition of antioxidants

such as ascorbic acid and N-acetylcysteine (NAC) inhibited the production of

DNA strand breaks, suggesting that ROS contributed to the breaks.

DNA fragmentation in Freshwater Cyanobacteria Nostoc spongiaeform and

Marine Phormidium corium due to high light mediated ROS production was

reported by Bhandari and Sharma (2006). Al at high concentrations in the range

0.5–2.0 mM is known to inhibit root growth in Arabidopsis through DNA damage

coupled with arrest of cell cycle in G2 stage (Rounds and Larsen, 2008). The

study conducted by Achary and Panda (2010) highlighted the dual role of Al

triggered ROS, which at low concentrations (1–10 µM) induced adaptive response

Chapter 1

Page 40

conferring genomic protection and at high concentrations induced DNA damage

in root cells that upheld the concept of biphasic (hormetic) dose response.

Desai et al., (2006) evaluated the DNA damaging potential of Cd in laboratory

cultures of phytoplankton species Chaetoceros tenuissimus, isolated from the

coastal waters by using Comet assay

1.1.4 Defense Mechanisms in Cyanobacteria against Oxidative Stress

In most of the organisms, cells are equipped with two types of antioxidant

systems; enzymatic and non-enzymatic to avoid damage caused by ROS.

1.1.4.1 Enzymatic Antioxidants

The enzymatic defenses include antioxidant enzymes such as peroxidases (POD;

EC 1.11.1.7) superoxide dismutases (SOD; EC 1.15.1.1) catalase (CAT, EC

1.11.1.16) and the major ROS scavenging system depends on the detoxification

mechanism which occurs as a result of the sequential and simultaneous action of

these enzymes. Thus, they constitute the first line of defense against reactive

oxygen species (ROS) and The activities of enzymes like ascorbate peroxidase

(APX, EC 1.11.1.11), guaiacol peroxidase (GPX, EC 1.11.1.7),

monodehydroascorbate reductase (MDAR, EC 1.6.5.4), dehydroascorbate

reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) are

also reported to increase under metal stress clearly deciphering their antioxidant

potential. Several studies have reported the implication of SODs in protective

processes in cyanobacteria exposed to a wide array of stresses. The first

Chapter 1

Page 41

implication on the protective role of cyanobacterial SOD in photo-oxidative

damage was shown in Anacystis nidulans (Herbert et al., 1992) Unlike other

living organisms, cyanobacteria possess multiple isoforms of SOD and are well

documented for its ability to maintain the antioxidant levels by releasing H2O2

into the environment (Patterson and Myers 1973, Morales et al., 1992; Kalavathi

et al., 2000), SOD, being the prime armory for the release of H2O2. In the

unicellular cyanobacterium Synechococcus PCC 7942, a sodB mutant, impaired in

the synthesis of iron SOD, was much more sensitive to oxygen and light than the

wild-type strain, suggesting a protective role of this SOD against damage to

photosystems, particularly PSI (Herbert et al., 1992). In the heterocystous strain

Anabaena cylindrica, SOD activity was been detected in both vegetative cells and

heterocysts. It has been assumed that it could be involved in the protection of the

proton donating systems of nitrogen fixation (Henry et al., 1978). In another

heterocyst-forming strain Anabaena PCC 7120, a MnSOD was suggested to be

involved in acclimation of this strain to high light (Zhao et al., 2007a, b). A two-

fold increase in SOD activity of dye treated L. valderiana BDU20041 suggested

the role of SOD in alleviating stress by Priya et al. (2010). Increased activity of

SOD was found in many organisms exposed to metal stress (Rijstenbil et al.,

1994; Okamoto et al., 2001a). It increased in Scenedesmus bijugatus exposed to

different Cu concentrations (Nagalakshmi and Prasad, 2001), in Spirulina

platensis-S5 exposed to heavy metals like Pb. Cu and Zn (Chaudhary et al.,

2006). Nearly 2.5 folds increase in SOD activity and 1.4 folds increase in GS

Chapter 1

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activity were observed at FeSO4 100 μM compared with control as reported by

Padmapriya and Anand (2010).

Several recent studies have helped in the understanding of the catalytic

mechanism of the bi functional catalase KatG from Synechocystis PCC 6803

(Smulevich et al., 2006). The function of this catalase in vivo has been studied by

analyzing the phenotype of a katG mutant. The data obtained suggested the

protective role of this enzyme against exogenous H2O2, and the involvement of

other peroxidases in coping with ROS produced inside the cells (Tichy and

Vermaas, 1999).

Ascorbate peroxidases (APX) also play a crucial role in H2O2 detoxification in

plants (Asada, 1999). These enzymes reduce H2O2 to monodehydroascorbate and

water using ascorbate as the specific electron donor. Monodehydroascorbate

spontaneously generates ascorbate and dehydroascorbate. Dehydroascorbate

reductase uses glutathione to reduce dehydroascorbate to ascorbate (Shigeoka et

al., 2002). Oxidized glutathione is then regenerated by NADPH-glutathione

reductase. This emphasizes the role of the ascorbate–glutathione cycle in the

response of plants to oxidative stress. In spite of a low level of ascorbate in

cyanobacteria, ascorbate peroxidase-like activities have been reported for Nostoc

muscorum PCC 7119 and for Synechococcus PCC 6311; and in Synechococcus

PCC 7942 dehydroascorbate reductase and glutathione reductase were involved in

the regeneration of ascorbate and glutathione, respectively (Tel-Or et al., 1985,

1986; Rozen et al., 1992). However, further biochemical characterization and

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overall genetic evidence is needed to prove the implication of these enzymes as

antioxidants in cyanobacteria.

There are also many reports available on the inhibition of enzyme activities by

heavy metals. Cd treatment decreased the chlorophyll and heme levels of

germinating mung bean seedlings by induction of lipoxgenase with the

simultaneous inhibition of the antioxidative enzyme SOD and CAT

(Somashekaraiah et al., 1992). Such inhibition results from binding of the metal

to the important sulfhydryl groups of enzymes, which exacerbates the phytotoxic

action of metals (Assche and Clijsters, 1990). Cu is known to interfere with

oxidative enzymes in oat leaves (Luna et al., 1994).

Non essential and non heavy metal like Al may also like other abiotic stress

disturb the redox homeostasis resulting in oxidative stress. Changes in their

activity of antioxidant enzymes and amounts have been identified as an indicator

of a redox status change under Al stress (Jan et al., 2001; Tamas et al., 2003;

Meriga et al., 2004; Simonovicova et al., 2004; Ali et al., 2008). As expected, the

activities of enzymes such as SOD, APX, MDAR, DHAR, and GR and the

concentrations of antioxidant metabolites like ascorbate (AsA), dehydroascorbate

(DAsA), reduced glutathione (GSH) and oxidized glutathione (GSSG), the

activity of CAT an enzyme involved in scavenging the bulk H2O2 generated by

photorespiration) (Chen et al., 2005a) and the activity of Rubisco in ‗Cleopatra‘

tangerine, were increased by Al (Chen et al., 2005b).

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Similarly, exposure to Al generally increased the activities of CAT, APX and

GPX, in Populus tremuloides and Populus trichocarpa leaves (Naik et al., 2009).

Al, at 160 µM, increased the activities of SOD, total and cytosolic APX, MDAR,

DHAR and GR suggesting that SOD, PGX and cytosolic APX played a key role

in combating oxidative damage (Sharma and Dubey, 2007). In detached rice

leaves, Al increased the activities of APX, CAT and GPX, but decreased that of

SOD. In longan, Al-treated leaves had higher or similar protein-based activities of

GPX, APX and GR and fresh weight-based GSH concentration depending on Al

concentration applied, but decreased CAT activity and AsA concentration (Xiao

et al., 2003a). In another study, Xiao et al. (2005) showed that Al increased the

activity of glycolate oxidase (GO, EC 1.1.3.15; a key enzyme involved in

photorespiration) in longan leaves.

1.1.4.2 Non-enzymatic antioxidants

The accumulation of ROS can be largely prevented by non-enzymatic

antioxidants among which α-tocopherol and carotenoids are the most important in

phototrophs. Also, recently researchers have started focussing on the role of some

Low Molecular Weight antioxidants (LMWA) like proline, phenolics etc. in

mitigation of abiotic stress. The uniqueness of this system is its direct interaction

with ROS of various kinds and its provision of protection for biological targets.

Cyanobacteria possess a wide variety of carotenoids like myxoxanthophyll, β

carotene, and its derivatives (zeaxanthin, echinenone). These pigments dissipate

Chapter 1

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energy from photosensitized chlorophyll or from 1O2, and several studies

emphasized their antioxidative properties (Young and Frank, 1996). Zeaxanthin

has been shown to be particularly important for photoacclimation during UV-B

stress in Synechococcus PCC 7942 (Gotz et al., 1999). A mutant of Synechocystis

PCC 6803 deficient in zeaxanthin synthesis became more sensitive to high light

and oxidative-stress treatment than the wild type (Schafer et al., 2005). Another

study which established the role of carotenoids as antioxidant showed that the

carotenoid composition in four freshwater cyanobacteria in response to high

irradiances overproduced and the percent increase varied from one

cyanobacterium to another (Schagerl and Muller, 2006).

The role of phenolic compounds (PhC) in oxidative protection is also ambivalent.

PhC are low molecular weight antioxidants that exhibit antioxidative properties

due to the availability of their phenolic hydrogens. Moreover, phenolic

metabolites can participate in ROS scavenging by cooperation with antioxidative

enzymes, e.g. with peroxidases in H2O2 scavenging (Sgherri et al., 2003). They

are constitutively expressed in higher plants and can effectively prevent oxidative

stress caused by unfavorable environmental factors, e.g. low temperature (Janas et

al., 2002; Grace, 2005), pathogen infections (Treutter, 2006), and UV radiation

(Bieza and Lois, 2001), heavy metals such as Cu (Sgherri et al., 2003; Ali et al.,

2006; Gorecka et al., 2007; Kovacik and Repcak, 2007). Their up-regulation can

be correlated with increased ROS/NO content which can serve as a signal for the

enhancement of phenolic synthesis finally resulting in the decrease in the effect of

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stress (Babu et al., 2003; Beta et al, 2005; Olichenko and Zagoskina, 2005; Rice-

Evans et al, 1996).

PhC are derived mainly from trans-cinnamic acid, which is formed from L-

phenylalanine in a reaction catalyzed by L-phenylalanineammonia-lyase (PAL,

EC4.3.1.5). Notwithstanding its importance, information related to PAL activity

under heavy metal stress is scarce. The effect of Cu on PAL was studied in Panax

ginseng (Ali et al., 2006) and the influence of Cd on PAL activity has recently

been studied in fronds of Azolla imbricata (Dai et al., 2006). However, more

detailed studies focused on time and concentration dynamics are still lacking.

Another important mechanism by which many plants and algae respond to and

apparently detoxify toxic heavy metals is the production of proline (Delauney and

Verma, 1993; Schat et al., 1997; Shah and Dubey, 1998; Mehta and Gaur, 1999;

Verma, 1999). Proline synthesis has been implicated in the alleviation of

cytoplasmic acidosis and may maintain NADP/NADPH ratios at values

compatible with metabolism (Hare and Cress, 1997). The mechanisms by which

Proline reduces free radical damage include physical quenching of oxygen

singlets (Alia et al., 2001) and chemical reaction with hydroxyl radicals (Smirnoff

and Cumbes, 1989).

Many plants accumulate high concentrations of proline when treated with toxic

concentrations of heavy metals (Bassi and Sharma, 1993a, b; Costa and Morel,

1994; Schat et al., 1997). Siripornadulsil et al. (2002) proposed that Proline

Chapter 1

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reduces heavy metal stress by detoxification of free radicals produced in response

to metal poisoning. A few reports have also appeared on algae (Chang, 1991; Wu

et al., 1998). Proline also accumulated in the cyanobacterium S. plantensis-S5

treated with heavy metals like Pb, Cu and Zn. (Choudhary et al., 2006).

Proline accumulation in plants also takes place in response to other abiotic

stresses such as salinity (Lutts et al., 1996), drought (Bates et al., 1975; Aspinall

and Paleg, 1981; Ibarra- Caballero et al., 1988), and low and high temperature

(Chu et al., 1974; Naidu et al., 1991). A study conducted by Chris et al. (2006)

showed that Cylindrospermum cells pre-treated with proline recorded low levels

of H2O2 generation, lipid peroxidation and electrolyte leakage due to UV-B

exposure which reflects its protective potential.

Many researchers believe that proline accumulation is a symptom of injury which

does not confer tolerance against metal or other stresses (Bhaskaran et al., 1985;

Lutts et al., 1996). Schat et al. (1997) observed that metal-induced proline

accumulation did not occur until the damage had been caused and, consequently it

did not apparently prevent metal toxicity.

Another salient point is that the accumulation of Proline in stressed plants is

associated with reduced damage to membranes and proteins indicating a

relationship between lipid peroxidation and proline (Shah and Dubey, 1998;

Verma, 1999).

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However, it is evident that there is no clear consensus regarding the mechanism(s)

by which Pro reduces heavy metal stress. Free Proline has been proposed to act

as an osmoprotectant (Paleg et al., 1984; Delauney and Verma, 1993; Taylor,

1996), a protein stabilizer (Kuznetsov and Shevyakova, 1997; Shah and Dubey,

1998), a metal chelator and an inhibitor of lipid peroxidation (Mehta and Gaur,

1999), a hydroxyl radical scavenger and a singlet oxygen scavenger (Alia et al.,

2001).

Rapid catabolism of Proline upon relief of stress also may provide reducing

equivalents that support mitochondrial oxidative phosphorylation and the

generation of ATP for recovery from stress-induced damage (Hare and Cress,

1997).

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Fig 1.4 Defense mechanisms against oxidative stress

Several other resistance mechanisms also exist to lessen or prevent metal toxicity.

These include resistance to metals that are always toxic to the cell and serve no

beneficial role, such as Cd and Hg, and also includes resistance to metals such as

Cu, Fe, Zn which are toxic at high concentrations but are absolutely essential in

trace amounts (Silver and Wauderhaug, 1992). Such mechanism involves extra

cellular binding whereby cells synthesize and release organic materials that

chelate metals to reduce their bioavailability (Clarke et al., 1987), or the metal

ions may be bound to the outer cell surface. These complex forms are generally

more difficult to transport into the cell. Secondly, cells can increase the rate of

metal ion excretion using energy-driven efflux pumps (Siegel, 1998). A third

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method of resistance is through internal metal sequestration. This is one of the

most important mechanisms by which bacteria combat heavy metal exposure and

subsequent accumulation. In the prokaryotic cyanobacteria, the class II

Metallothionins (MTs) performs metal ion sequestration within the cell. Another

important resistance mechanism used by cells in response to a variety of

environmental stressors is the expression of heat shock genes. These proteins are

present in highly conserved forms in bacteria, plants, and animals.

1.1.4.3 Metal Chelation by stress proteins

GroEL

One of the most important heat shock proteins is GroEL. GroEL is a 58-kDa

protein that assembles into two stacked rings of seven subunits each with an

additional ring of seven 10-kDa GroES subunits. This complex has been shown to

renature proteins, making them again functional (Weissman, et al., 1996). Since

their major role is in assisting protein folding with the consumption of ATP,

GroEL and GroES are termed chaperonins. Chaperonins provide the kinetic

assistance to the process of folding of newly translated proteins or proteins

disrupted as a result of cellular stress (Xu et al., 1997). In the bacteria, the genes

for GroES and GroEL proteins are arranged into an operon (groESL) and

transcription is coordinately expressed by the use of specific stress sigma factors.

GroEL has been shown to be an essential component for maintaining viability

with changes in temperature (Webb et al., 1990). GroEL and GroES are essential

proteins for cellular growth and are always transcribed at baseline levels; only

Chapter 1

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under conditions of stress does the transcription rate increase. The study

conducted by Ybarra et al. (1999) focused on the cellular effects of exposure to

divalent metal cations and the roles of the specific resistance mechanism of MT

expression, and the expression of the stress protein GroEL in cyanobacterium

Synechococcus sp. Strain PCC 7942. It appears that the sequestration of these

metals by metallothioneins detoxifies them, thus decreasing their detrimental

cellular effects. They increase the transcription of groEL as a resistance

mechanism in response to the many types of cellular alterations resulting from

environmental contamination.

Fig 1.5 Hypothetical mechanistic model depicting the survival strategy of

A.doliolum under excess Cu. ‘X’ represent inhibition, and thick arrows

indicate enhancement (Bhargava et al., 2008)

Chapter 1

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Phytochelatins (PCn)

Production of (PCn) (g Glu-Cys)n Gly, (n=2–11) and their homologs differing in

the terminal amino acid is also a constitutive mechanism for coping with elevated

toxic metal concentrations (Rauser, 1995; Zenk, 1996). These thiol-containing

peptides are produced in cells of plants, algae and fungi exposed to some heavy

metals and metalloids like Cd, Cu, Pb, Zn, Hg, Ag, As (Zenk, 1996; Singh et al.,

1997; Ahner and Morel, 1999) that can exert toxic effects on living organisms.

Algae are able to bind high amounts of heavy metals from the surrounding water

(Revis et al., 1989), and they respond to the intracellularly accumulated metal by

synthesizing PCn of different chain-length (Gekeler et al., 1988; Ahner and

Morel, 1995; Pawlik-Skowron´ska, 2000). Mallick and Rai (1998) demonstrated

the production of a 3.3 KDa protein, rich in cadmium and –SH contents and

sensitive to buthionine-sulfoximine (BSO), in a diazotrophic cyanobacterium

Anabaena doliolum following 20 μM Cd exposure. The study proved that this

protein resemble the higher plant PCn and offers not only co-tolerance to different

heavy metals but also provide multiple tolerance to a host of environmental

stresses.

Metallothioneins (MT)

The entrance of certain metals into the nucleus can enhance the synthesis of RNA

that codes for MT. MT are peptides found mainly in the cytosol, lysosomes and in

the nucleus, low molecular weight peptides, high in the amino acid cysteine,

which contains a thiol group (-SH). The thiol group enables MTs to bind heavy

Chapter 1

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metals. MT can be induced by essential and non-essential metals in aquatic

organisms (mollusks, crustaceans). The MT induction leads to changes in several

biochemical processes that have the potential to be used as biomarkers of

exposure and evaluation of pollution in the marine environment. (Hamer, 1986)

Fig 1.6 General scheme of heavy metal detoxification and metal homeostasis

in microalgae mediated by PCn and MTs (Hirata et al., 2005)

Dehydrins

Dehydrins are group 2 (late embryogenesis abundant) LEA proteins which are

distributed not only in higher plants but also in algae, yeast, and cyanobacteria

(Close, 1996; Ingram and Bartels, 1996;Svensson et al., 2002). Drought, cold,

Chapter 1

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salinity, and the administration of abscisic acid (ABA), all promote the gene

expression and accumulation of dehydrin protein (Close,1997; Svensson et al.,

2002) It has recently been reported that transgenic tobacco expresses citrus

dehydrin with fewer peroxidized lipids than the wild type when incubated at low

temperatures(Hara et al., 2003). Citrus dehydrin shows scavenging activity

against hydroxyl radicals and peroxyl radicals (Hara et al., 2004). During the

process of scavenging by citrus dehydrin, Gly, Lys, and His residues are markedly

degraded, suggesting that the three are involved in the scavenging of radicals

(Hara et al., 2004). This antioxidative activity may be a crucial function of

dehydrin, because dehydrins are produced not only by cold but also by drought

and salinity, both of which are environmental stimuli which generate radicals in

plants (McKersie et al., 1993; Shen et al., 1997; Iturbe-Ormaetxe et al., 1998). It

is postulated that hydroxyl radicals, which are extremely cytotoxic, are generated

during the response to water stress in plants (Iturbe-Ormaetxe et al., 1998). The

hydroxyl radical is generated by the metal-catalysed Haber–Weiss reaction in

which transition metals, such as Fe and Cu, participate (Halliwell and Grootveld,

1988). In both transition states, i.e. as Fe2+

and Fe3+

or Cu+ and Cu

2+ , these metals

can form hydroxyl radicals by reacting with hydrogen peroxide or superoxide

anions. It is known that some antioxidative proteins, such as metallothionein,

ceruloplasmin, and serum albumin, bind metal ions to stabilize them (Soriani et

al., 1994; Vasak and Hasler, 2000; Kang et al., 2001; Akashi et al., 2004).Indeed,

it is reported that some dehydrins can bind metal ions (Svensson et al., 2000;

Kruger et al., 2002; Alsheikh et al., 2003; Herzer et al., 2003). Svensson et al.

Chapter 1

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(2000) noted that His residues in dehydrins could be involved in dehydrin-metal

binding, because some dehydrins are rich in His, which is one form of metal-

affinity residue.

Alterations in the proteome of Cyanobacteria under Stress

Cyanobacterial adaptation to stress is coupled with profound changes in proteome

repertoire. hence they are known to adapt to environmental stresses by suitably

modifying their proteome. Since proteins are directly involved in stress responses,

proteomic studies can unravel the possible relationships between protein

abundance and stress acclimation. One-dimensional gel electrophoresis gives a

first hand information regarding the alterations in the protein profile of stressed

organisms. Further 2DE (two-dimensional gel electrophoresis) followed by

MALDI-MS analysis has emerged as a powerful technique for resolving the

proteome of different organisms. Over the past years, the proteomic approach has

been applied to analyze the proteins involved in stress responses in cyanobacteria,

such as Cu (Bhargava et al., 2008), salinity (Fulda et al., 2006; Srivastava et al.,

2008) heat and UV-B (Suzuki et al., 2006; Mishra et al., 2009; Gao et al., 2009),

butachlor (Kumar et al., 2009) and Fe starvation (Narayan et al., 2011)

Apte and Bhagwat, (1989) successfully analysed the stress stimulon of salinity-

tolerant Anabaena sp. L31 subjected to heat, salinity and osmotic stress and

demonstrated induction of two different sets of polypeptides. One set with four

polypeptides was common for all the three stresses studied, while the other set of

four polypeptides was unique to heat stress only. Cd-stress stimulon in

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Escherichia coli8 and UV-B stress stimulon in desiccation tolerant Nostoc

commune (Ehling-Schulz, 2002) have also been successfully investigated using

proteomics. Further, Sazuka (Sazuka, 2003) analysed the proteome of Anabaena

sp.PCC 7120 using 2DE and characterized 123 most abundant proteins.

Fig 1.1 Proteomic analyses of the response of cyanobacteria to different stress

conditions. (Castielli et al., 2009)

1.1.5 Stepwise exposure of cyanobacteria to increasing concentration of metal

and the concept of adaptation

A critical perusal of the literature revealed alterations in the growth, and

physiological and biochemical processes of cyanobacteria exposed to heavy

metals (Prasad et al., 1991; Rai et al., 1995; Bhargava et al., 2008),

Chapter 1

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temperature (Mishra et al.,2005; Srivastava et al., 2006) and salinity (Srivastava

et al., 2005, 2006) as a result of which cyanobacteria ought to acclimate these

stresses for their survival. It is relevant to state that reports are available in the

literature on copper-acclimated pea (Palma et al., 1987), mustard (Wang et

al., 2004), yeast (Miura et al., 2002) and algae such as A. doliolum (Rai et al.,

1991) and Enteromorpha compressa (Ratkevicius et al., 2003). Moreover it is

also well established in literature that despite the proven toxicity prolonged and

repeated exposure of cyanobacteria to metals like Cu has been found to induce

physiological tolerance (Rai et al., 1991; Shavyrina et al., 2001).

In most cases mutations based genetic variability is the result of the natural

selection which leads to evolution but the occurrence of adaptive mutations in

bacteria and yeast (Foster, 2000; Heidenreich, 2007) added a new view on

evolutionary biology. In particular, there is great interest in understanding

whether adaptive mutations play any evolutionary role in adaptative processes

(Foster, 2000; Perfeito et al., 2007). However, it is supposed that they can provide

a valuable supplement to the conventional process of mutation and selection only

when the selective stress is non-lethal (Wintersberger, 1991).

Chapter 1

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1.2 Hypothesis

In the past few years environment become more unstable due to the continuous

contribution of metals, variable salinity and fluctuations in fresh water

availability caused by anthropogenic activities such as industrialization and

urbanization. These activities mobilising the toxic metals like cadmium (Cd),

Nickel (Ni), Palladium (Pd) and Mercury (Hg) into the ecosystems which

ultimately have created significant ecological misbalance. Among various types

of ecosystems, aquatic ecosystems play an important role due to the presence of

primary producer ―Algae‖ including cyanobacteria. Cyanobacteria are a group

of ubiquitous, photosynthetic prokaryotes which perform two key biological

processes such as oxygenic photosynthesis and nitrogen fixation in same

filaments. They are ecologically important group of eubacteria that evolved to

compete in a wide range of habitats. They have evolved under anoxic

conditions and are well adapted to environmental stress including exposure to

UV, high solar radiation and temperatures, scarce and abundant nutrients (Zhu,

2001). Adaptation in such harsh environment has favored the dominance of

cyanobacteria in many aquatic habitats, from freshwater to marine ecosystems.

Heavy metals are the major environmental pollutants which are spilled to soil

and aquatic ecosystems through several agencies. Being non-degradable, they

get readily incorporated into the biogenic materials particularly phytoplankton.

Under these conditions, algae and cyanobacteria must be able to respond rapidly

to unpredictable changes which probably lead to a variable degree of oxidative

stress. It is well-known that reactive oxygen species (ROS) are produced in

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normal metabolic processes in all aerobic organisms (Mittler, 2002, Asada and

Takahashi, 1987) and also well established is their implication as key molecules

in pathogen defence, programmed cell death, abiotic stress responses and

systemic signaling (Desikan et al., 2001; Mittler, 2002). However, several stress

conditions (metal pollution, salt stress, chilling, UV radiation, pathogen attack,

etc.) can unbalance the steady-state level of ROS production (Foyer et al.,

1997). These ROS include the superoxide radical (O2•−

), hydroxyl radical (•OH)

and hydrogen peroxide (H2O2), which are produced during electron transport

activities in the cell membrane as well as by a number of metabolic pathways

(Shi et al., 2006). ROS accumulation induces oxidative processes such as

membrane lipid peroxidation, protein oxidation, enzyme inhibition and DNA

and RNA damage, resulting in cell damage and, eventually, cell death (Dat et

al., 2000; Hammond-Kosack and Jones, 1996). Afterwards, the ROS could be

detoxified by efficient antioxidant defense mechanisms, comprising both

enzymatic components, such as ascorbate peroxidase (APX), catalase (CAT),

superoxide dismutase (SOD) or guaiacol peroxidase (POD); as well as non-

enzymatic components, such as ascorbic acid, proline or glutathione and

expression of stress specific proteins (Arrigoni and De Tullio, 2002).

Understanding the fundamental physicochemical mechanisms of metal bio-

uptake by cyanobacteria in natural systems is a step towards identifying under

what conditions cyanobacterial growth is favoured and to ascertain the

mechanisms by which the excess and type of metal is detoxified making

cyanobacteria an excellent tool for toxicity assay. Our proposed hypothesis is

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that exposure of cyanobacteria to different metals leads to metal specific stress

and subsequently to expression of differential response to avert the toxicity

thereby enhancing the survival of the organisms.

Our approach includes the following major goals:-

Objectives of the proposed work:

1. Survival, Growth and Photosynthetic responses of cyanobacterium

N.muscorum to metal stress.

2. Evaluation of possible protective mechanisms of N. muscorum exposed to

Al, Cu and Cd

3. Evaluation of metal-induced molecular modulations in protein, DNA and

lipid in the survival of N. muscorum

4. Role of proline in modulation of Al-induced stress responsive proteins:

relationship between adaptation and proline accumulation

1.3 Significance of work

Al-induced stress in N.muscorum is probably dealt with a different mechanism(s)

as compared to the Cu and Cd. Largely unknown nature and role of these Al-

induced proteins in N.muscorum certainly added sufficient curiosity to further

isolate, identify and characterize their physical, chemical and biological properties

which will likely to enhance our understanding of Al-induced tolerance in

N.muscorum.

Thus understanding the molecular basis for these mechanisms will be an

important aspect in developing bio removal tools to clean the contaminated water

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bodies. We believe that only through compound studies one can gain the insight

needed to make predictive statements regarding the effect of metal toxicants on

cyanobacteria from the aquatic ecosystems.