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Review of literature features of mangal waterlogged soil typical mangrove habitats are periodically inundated by


2. REVIEW OF LITERATURE

2.1. Features of Mangal

2.1.1. Waterlogged soil

Typical mangrove habitats are periodically inundated by the tides. Therefore they grow in soil that is more or less permanently water logged and in water whose salinity fluctuates and may be as high as that of the open sea. Generally the under ground tissues of any plant require oxygen (02) for respiration. In soils, which are not water logged, gas diffusion between soil particles can supply this need. In a water logged soil, the spaces between soil particles are filled with water. Even when water is saturated with oxygen, its oxygen concentration is far below that of air, and the diffusion rate of oxygen through water is roughly 10000 times less than through air (Ball, 1988a). Therefore water logged soils are low in oxygen. When the oxygen movement in to soil is severely limited, the oxygen that is present in the soil is soon depleted by the aerobic respiration of soil bacteria. There after, anaerobic activity takes over. The result is that mangrove soils are often virtually anoxic. Mangrove trees have adapted to survive in such unpromising surroundings. The most striking adaptations are various forms of aerial roots. In water-logged soils, special aerating devices are required since the underground roots are in the permanently hypoxic, or even anoxic, environment. The functional vertical columns of the roots have the role of supplying 02 to the underground roots. Air passes in to the vertical columns through numerous pores, which are particularly abundant close to the point at

10

which the column root enters the soil surface. Roots entering the soil are largely composed of aerenchyma tissue, honeycomb like with air spaces which run longitudinally down to the aerial root axis. Root architecture differs according to species but also to some extent depends on the prevailing conditions. Mangroves whose roots are regularly inundated by the tide have abundant pneumatophores. On the landward side a few rather stunted trees have contrived to establish themselves in apparently dry sand, where they seem to survive on underground seepage of fresh water. The surface soil is essentially sandy through which air can readily penetrate. In such case there is no need for special aerating structures: pneumatophores are entirely absent. The rooting system of mangroves also has the function of anchorage. Mangrove roots take up water much less freely than those of conventional plants. The objective of this is to assimilate carbon in photosynthesis which cannot be done without expenditure of water. Consequently, a greater relative root mass is needed to satisfy the demand for water. In Avicenia the root: shoot ratio is relatively high, and it increase with increasing salinity (Ball, 1988b; Saintilan, 1997). In a mangrove forest, water is not in short supply but its acquisition is costly because of the problems of copying with salt. Although mangrove plants are surrounded by water, they cannot afford to be extravagant in its use. In mangroves the ratio of water used and the carbon assimilated is higher than non-mangrove species, particularly in salt tolerant species (Hogarth, 1999). The entry of CO2 and the transpiration of water occurred through the leaf stomata. At high soil salinities, stomata! conductance and the passage of gases through stomata are reduced. This 11

conserves water by reducing transpiration, but also reduces CO2 uptake and growth. The balance between the two is such that, except under extreme condition, just sufficient water is expended to maintain the carbon assimilation rate very near the photosynthetic capacity of the leaf. Therefore mangrove must achieve a balance between minimizing water expenditure, holding down leaf temperature and maximizing CO2 acquisition and growth (Ball and Pidsley, 1995).

2.1.2. Coping with salt

Mangroves typically grow in an environment whose salinity is between that of fresh water and sea water. In some circumstances, mangroves can survive in hypersaline conditions, and the problem of water acquisition is correspondingly worse. For instance Indus delta of Pakistan, evaporation raises the salinity to twice that of the sea (Hogarth, 1999). Mangroves exhibit a variety of means to cope with this unpleasant environment. The principal, mechanisms are exclusion of salt by the roots, tolerance of high tissue salt concentration (salt accumulation) and elimination of excess salt by secretion. The interplay between these is complex, and it may vary between taxa. In Avicennia, 90% of the salt is excluded at the root surface, rising to 97% as the salinity of the environment increases (Tomlinson, 1986). Although the mechanism of salt exclusion is not understood clearly it appears to be a physical one. Negative hydrostatic pressure is generated with in the plant by transpiration pressures, this is sufficient to overcome the negative osmotic pressure in the environment of

12

the roots. Water is therefore drawn in and unwanted ions and other substances such as dye molecules are excluded (Moon et aI., 1986). In several mangrove species, viz. Avicennia, Rhizophora, Sonneratia and Xylocarpus, sodium chloride is deposited in the bark of stem and root. The deciduous mangrove Xylocarpus and Excoecaria appear to dump excess salt in senescent leaves for a new fruiting and growing season (Hutchings and Saenger, 1987). It is also reported that the lower leaf surface of Avicennia is densely covered with hairs, which raise the secreted droplets of salty water away from the leaf surface, preventing the osmotic withdrawal of water from the leaf tissues (Osborne and Berjak, 1997). The glands of Avicennia appeared to be formed only in response to saline conditions while in Aegiceras, and most other gland bearing species, they are present regardless of the environmental salinity. The secretary cells of the glands are packed with mitochondria suggesting the intense metabolic activity. Thus exclusion, tolerance and secretion are used with different emphasis by different species, and within a species under different environmental conditions (Hogarth, 1999). Table 2 summarizes the known occurrence of these mechanisms i.e., exclusion, accumulation and secretion in a range of mangrove species.

2.1.3. Inorganic nutrients

In addition to water, plants also require inadequate supply of mineral nutrients. The most important of these are nitrogen and phosphorus, in the form of inorganic nitrate and phosphate. Possible sources of inorganic nutrients are rainfall, fresh water from rivers or as runoff from land, tide borne soluble or particle bound nutrients, the bacterial fixation of atmospheric

13

SI. No.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Table 2.

Species Exclude Secrete Accumulate

Acanthus + Aegia!itis + + Aegiceras + + Avicennia + + + Bruguiera + Ceriops + Excoecaria + Laguncularia + Osbornia + + Rhizophora + + Sonneratia + + + Xy!ocarpus +

Mechanism of coping with salt and their known distribution in a range of mangrove species.

nitrogen and release by microbial decomposition of organic material. Overall levels of these elements are affected by a complex and shifting balance between different sources: their actual availability to the mangroves depends usually on the nature of the mangrove soil, and on microbial activity within it. Mangrove trees and their soil have a complicated relationship. Potentially, mangroves also have a nutrient source in regular tidal inundations.

2.2. Salinity

Many of the commonly found ionic molecules in soil and H20 are intact, toxic to living cells at relatively low concentration. Arid regions of the world are associated with soil which frequently contain high levels of ionic salts such as sodium chloride (NaCl), sodium sulphate (Na2So4) and calcium chloride (CaCb). These salts are normally contained in soils, but in regions of moderate to high rainfall, they are leached from the soil into the ground water and ultimately the ocean, which is therefore quiet salty. Wetter regions of the world are associated with acidic soil. Rainwater contains dissolved CO2, which is carbonic acid. H + ions are readily produced by the dissociation of carbonic acid. The chemical activity of H + is such as that it displaces other cations from soil particles. The other cations including useful ions such as calcium (Ca ++ ), magnesium (Mg + +) and amonium (NH/) are leached into the ground water leaving the soil acidic and nutrient poor. In addition toxic metals such as aluminum (Al), which are in a precipitated form in the soil, are brought into the soil solution in acidic soils. Thus, wet and dry soil has salinity problems (Greenway and Munns, 1980).

14

'' . ; \ ,;;

In terms of plant production, salinity can be defined as the excessive concentration of soluble salts in soil. If the salt concentration is high enough to lower the water potential (\jJ) appreciably, the stress will be called as salt stress (0.5 to 1 bar). If the salt concentration (acidic/basic) is not high enough to lower \jJ appreciably the stress will be called as ion stress (Bohnert; et aI., 1995). It is obvious that salt stress in soil varies considerably with soil moisture level. It is evident from the reviews that saline stress in plant growth has attracted the attention of several researchers and research organizations for decades ago (Takada, 1954; Ungar, 1962; Kinzel, 1963; Weissenbock, 1969; Waisel, 1972; Strongonov, 1973; Poljakoff-Mayber and Gate, 1975; Greenway and Munns, 1980; Bohnert et aI., 1995; Marcum, 1999; Agastian et aI., 2000; Hamilton et aI., 2001; Mac Farlane, 2001 and Mimura, 2003). Generally the nature of salt stress in plants depends on the habitat and environmental factors (Bohnert et aI., 1995). The physiological effects of salt on plants are diverse and they are clearly defined based on their ecological adaptations. From the earlier studies it was clear that the accumulation of salt in the vacuole does not physiologically interfere with plant function, whereas accumulation in the cytoplasm has varied, deleterious effect. The cytoplasm of a tolerant plant can adapt to high salt concentration or can exclude toxic salts. On the basis of level of salt concentration two groups of plants can be distinguished broadly: salt lovers otherwise called halophytes are capable of survival on salt concentration exceeding 300 mM (Flowers et aI., 1977). Conversely sugar lovers or glycophytes cannot complete a life cycle at salt concentration exceeding 300 mM. At a salt concentration of 20 to 200 mM there is an overlap of growth responses between the two groups. 15

There is a direct and inseparable relation between the salt and H20 stress. Since the addition of a salt to H20 lowers its osmotic potential, the salt stress must expose the plant to a secondary physiological drought stress which otherwise called water stress (Bray, 1993). Therefore, if a plant is transferred from a lower salt to high salt medium, it is immediately subjected as osmotic dehydration. This osmotic dehydration may be the immediate cause of salt injury, such as depression of growth, decrease in transpiration and stomatal imbalance in opening and closing (Casas et al., 1991; Ball and Pidsley, 1995; Ball, 1998; Tezara et ai., 2003). The decreased growth due to salinization has been explained by a suppression of nutrient absorption due to uptake of sodium chloride (NaCI) in competition with nutrient ions (Allen et ai., 1995). Addition of NaCI to the root medium of barley plant markedly increases leaf RNase activity (Niu et ai., 1995). Since this rise in RNase activity is associated with water stress injury, it indicates that salt stress is inducing a secondary water stress injury. Most of the salt stresses in plants is due to sodium salt particularly NaCI. Under saline conditions the cells of halophytes accumulates sodium ions and Chloride ions (CI -) in excess. The Na+ salt injuries are cessation of growth or by an actual killing of the tissues in the form of necrosis or marginal burn, followed by a loss of turgor, falling of leaves and finally death of the plant (Cheeseman, 1988; Adams et ai., 1992; Niu et ai., 1995; Amtmann and Sanders, 1999). Primary indirect salt injury caused by NaCI are inhibition of mitotic activity, delay in flower emergence, hastened the ripening of fruit and defoliation of leaves (Naidoo and Rughunanan, 1990; Reddy et ai., 1992; Arbona et aI., 2003; Lee

16

et al., 2003). Similarly NaCl depress photosynthesis, reduce chlorophyll content, and inhibit Co2 uptake (Flowers, 197 4; Al-zaharani and Hajar; 1998; Rai et al., 2003). Apart from sodium salt, the calcium (Ca++) salt stress also injures the plants primarily because of the accompanying bicarbonate. When large quantities of calcium are taken up by the plants they can usually be precipitated in the cell sap as oxalates or carbonates or neutralized in the cell sap as malates or citrates. Based on the physiological availability of Ca++ in contrast to the above ecological classification as halophytes and glycophytes plants are grouped as calciotrophs or calciophiles and calciophobes (calciofuges). Calciophiles are those plants that contain large quantities of soluble calcium in the cell sap · where as calciophobes contain very little soluble calcium in its cell sap but more or less large quantities of insoluble calcium (Knight et al., 1997; Sanders, 2000; Zhu, 2001 ). The salt stress led to the accumulation of intermediates of the glycolate pathway, and increased levels of reducing sugars but decreased amounts of sucrose and starch (Popp, 1995; Ueda et al., 2003). No salt decreased the respiration rate of many crop plants (Wang et al., 2001 ). Conversely, the mitochondria were inhibited by concentrations of NaCl above 125 mM (Storey et al., 1993). NaCl decreases protein synthesis and increases its hydrolysis in many crop plants. A stimulation of protein synthesis has also been found (Colmer et al., 1996; Bouchereau et al., 1999). In grape leaves, salinity disturbed nucleic acid metabolism leads to growth inhibition (Tai, 1977). A comparison of enzymes, such as amylase, invertase in leaves of salt tolerant and salt sensitive plants has shown considerable variation in the effect of salt (Guo et al., 2001 ). 17

~ l. '7losUP~·'.r ..,,- , .' Activity of invertase was found reduced with salinity in salt sensitive plants whereas activity of the enzyme did not change in salt tolerant species. Direct exposure to NaCI depending on the extent of salt accumulation by the cytoplasm affects several enzymes adversely, malic dehydrogenase, aspartate transaminase and isocitrate dehydrogenase isolated from halophytic plants showed NaCI sensitivity like those from salt sensitive plants (Binzel et aI., 1985; Tropin et aI., 2003). Peroxisomal glycolate oxidase appeared to be relatively insensitive under in vitro conditions. High molecular weight proteins remained more active at higher salt concentrations than its lower molecular weight forms (Ford, 1984). Two enzymes consistently increase on salination, nitrate reductase and ribonuclease (Tal, 1977). Salts have two antagonistic effects on proteins: (i) they tend to break the electrostatic bonds and (ii) to increase the hydrophobic interactions (Melander and Horvath, 1977). There is therefore, no basic reason for expecting a general increase or decrease in enzyme activity. On the contrary, both effects may be expected depending on the specific structural characteristics of the protein. Large increase in peroxidase activity due to salt stressed leaves may have played a role in the oxidation of the accumulated substances, leading to melanin formation from tyrosine in the necrotic areas. Catalase activity also increased indicating a toxic accumulation of H20 2 (Mittova et aI., 2003; Vattanaviboon et aI., 2003). Apart from these enzymic variations, plants also exhibit certain morphological and anatomical features in response to salinity (Ortega and Taleisnik, 2003). These include: increase in leaf succulence; expansion of leaf lamina; changes in number and size of stomata; thickening

18

of cuticle; extensive development of tyloses; increased lignification and the presence of salt glands. The mechanism of salt transport to salt glands has been reviewed extensively by various workers (Matoh et aI., 1987; Ball, 1988b; Marcum, 1994). Cells comprising the salt glands possess dense cytoplasm, many mitochondria, large nucleus, small vacuoles and ribosomes. The salt inhibits the activities of some enzymes and stimulates others, particularly hydrolases. Seven enzymes from Halobacterium cutirubrum require high salt concentrations for both activity and stability. Magnesium chloride (MgCI2) and calcium chloride (CaCh) are sometimes even better activators than K+ or Na+ salts (Maathuis and Amtman, 1999; Arbona et aL, 2003). The cations presumably act by neutralizing negative electrostatic charges surrounding the binding site that prevent approach of the substrate; the anions, by affecting the conformation of the enzyme, thus altering the accessibility of the binding site to the bulk solvent (Martinoia et aI., 1986; Hasegawa et aI., 2000a). In the case of malate dehydrogenase from the extremely halophilic bacteria from the Dead Sea, inactivation at low salt concentrations is ascribed to a dissociation of the enzyme. Reactivation occurs when high salt concentration leads to reassociation (Galinski, 1993; Burg, 2003). In spite of positive results, it has been shown that the enzymes of halophytes are inhibited by much lower ion concentrations than those expected in the cell, and in fact, may not differ from their counterparts in glycophytes with respect to their sensitivity to salts in vitro. For instance, in the obligate halophyte, Suaeda maritima, the enzyme glutamate dehydrogenase is activated by 25 mM NaCI and inhibited by concentrations 19

greater than 100 mM, by affecting synthesis of the enzyme (Ramanjulu and Sudhakar, 2001). Plants that possess C4 photosynthesis commonly seem to require NaCI. Even in the same plant, NaCI appears to switch the plant from C3 to C4 photosynthesis due apparently by activation of phosphoenol pyruvic carboxylase by CI - (Beer et aI., 1975; Furbank and Taylor, 1995). Others have failed to confirm this relation, supposedly because the C3 path occurs only in the nearly complete absence of NaCI (Robinson, 1985; Ball and Munns, 1992; Arp et aI., 1993). Perhaps because of these switch mechanisms, salinization decreases the organic acid content of cotton, and increases it in an obligate halophyte (Lu et aI., 2003). The primary direct strain produced by a sudden salt shock is injurious via membrane damage. Tolerance involves membrane properties. In higher plants, mitochondria are salt restricted, chloroplasts are severely affected. There was a causative connection between the efflux of K+, and the change in the fine structure of the chloroplasts (Lu and Vonshak, 2002; Yamane et aI., 2003). The existence of both salt avoidance and salt tolerance mechanisms leads to a paradoxical relation between resistant and sensitive plant. Since avoidance prevents salt accumulation, a salt-sensitive plant relative to the salt avoiding plant would be one that permits the accumulation of salt in its cells (Breckle, 1974a; Bohnert and Jensen, 1996; Nuccio et aI., 1999; Loelock and Feller; 2003). The accumulation of salt both by a nonavoiding salt-sensitive plant and by a tolerant, salt-resistant plant may mean that the salt accumulates in the cytoplasm of the sensitive plant but only in the vacuole of the resistant plant. In may also, however, involve accumulation in the vacuole of both but only

20

the salt-tolerant one may be capable of compensating for this intracellular osmotic strain by a sufficient accumulation of organic solutes in the cytoplasm to maintain normal cell turgor and growth (Loewus and Loewus, 1982; Mc cue and Hanson, 1990; Rhodes and Hanson, 1993; AI- Zaharani and Hajar, 1998 Nuccio et aI., 1999; Golldack et aI., 2003). 2.2.1. Accumulation of organic solutes

Accumulation of several types of organic solutes has been suggested to play a role in osmoregulation because of the cellular compatibility with other macromolecules. These solutes are generally produced in plants under salt 0" water stress. Osmotic protectants are highly soluble compounds that carry no net charge at pH and are non toxic at high concentrations. They serve to raise osmotic pressure in the cytoplasm and can also stabilize proteins and membranes when salt levels or temperatures are untolerable. Thus they play important roles in the adaptations of cells to various adverse environmental conditions (Bartels and Nelson, 1994; Szegletes et aI., 2000; Silveira et aI., 2003). The most important of these organic solutes are sugars, free aminoacids, methyl amines and polyhydric alcohols (Tarczynski et aI., 1993; Mergulhao et aI., 2002; Chattopadhayay et al; 2003., Waditee et aI., 2003). These solutes, unlike salts, do not inhibit in vitro enzymes even at a concentration of 500 mM (Abebe et aI., 2003). Accumulation of organic solutes seems to occur in association with reduction in protein and polysaccharide synthesis and subsequent growth inhibition (Egan et aI., 2001). Whether organic solutes actually protect the plant from 21

salt stress or they are merely the result of such stress is still a mootpoint and requires further research. It is also unclear whether salt stress plants exclude toxic ions from the cytoplasm and rely on organic solutes for osmotic balance (Hanson et al., 1994; Blunden et al., 2001; Sakamoto and Murata, 2001, 2002; Munns, 2002). Free aminoacids such as praline are important regulators of cellular osmotic pressure (Handa et al., 1986; Delauney and Verma, 1993 Phutela et al., 2000). It has also been advocated as a cytosolute in halophytes however the accumulation of this compound is a near-universal response to water stress and may indeed be a measure of internal water stress. Nevertheless praline may be the major cytosolute in the salt adaptation of some plants. (Storey and Jones, 1977; Yoshiba et al., 1997; Lutts eta!., 1999;; AI-Khayri, 2002 Arvin and Kazemi - Pour, 2002; Tarakcioglu and lnal, 2002; Mergulhao et al., 2002). Water stress is one of the most common environmental limitations for plant growth and it causes major alterations in various metabolic processes of plants. The cellular response to water stress triggered the accumulation of an osmotically active compound called praline in cell stem. This osmolyte regulate the turgidity as well as activates the absorption of additional water from the soil. Praline is one of the most important osmolyte that accumulates in plants subjected to drought and salt stress. Its role as a potent osmoprotectant was first demonstrated by the increased osmotolerant characterized in a mutant Salmonella species (Csonka, 1989). A series of studies regarding the praline metabolism in higher plants conclusively established this iminoacid as an osmoprotectant that enhance tolerance in salt stress (Garcia- Carmona et al., 22

1988; Yang et al., 1996; Mattioni et al., 1997; Gadallah et al., 1999 Makela et al., 1999). The stimulation of praline biosynthesis under salt and water stress was shown to be associated with an increase of mRNA levels. Several studies have indicated that pyrroline 5 - carboxylate synthetase is a critical enzyme in praline biosynthesis. Another class of organic osmoregulators is the methyl amines in the salt stressed plants. Glycine betaine (GB) is one which studied extensively as an osmoregulator in halophytic plants (Storey and Jones, 1977; Nakamura et al; 1996., Wang et al., 2001 Sullpice et al., 2002). This cytosolute is found in higher amounts in salt tolerant species, and in low amounts in salt sensitive plants (Waditee et al., 2003). However its level showed considerable increase in salt sensitive spinach leaves under salt stress. Localization of glycine betaine in cytoplasm was demonstrated by sub- cellular fractionation and histochemical studies (Hall et al., 1978). Accumulation of glycine betaine occurs predominantly under gradual stress, whereas praline accumulates under highly damaging lethal salt levels (Makela et al., 2000). Unlike praline, glycine betaine not degraded rapidly. GB occurs in diverse marine algae and most flowering plant families (Rhodes and Hanson, 1993). Its synthesis has been studied mainly in species of Chenopodiaceae and Gramineae. In these cases, GB is synthesized via a two step process i.e., choline to betaine aldehyde is mediated by choline monooxygenase (CMO), an unusual ferrodoxin dependent monooxygenase, which has been characterized and cloned (Rathinasabapathi et al., 1994; 23

Russell et al., 1998). The second step of GB synthesis is catalyzed by betaine aldehyde dehydrogenase (BADH) is a dimer of identical 54 kD subunits (Nakamura et al., 1996). BADH is induced by osmotic stress (Rhodes and Hanson, 1993). GB accumulation has long been a target for engineering stress resistance (le Rudulier et al., 1984 ). The idea that introducing the GB pathway into plants that lack it will enhance their stress tolerance is based both on comparative physiology and on genetic evidence from a mutation in maize that abolishes GB synthesis and reduces salt and heat tolerance (Yancey, 1994; Saneoka et al., 1995; Yang et al., 1996). Wide crossing work on Lophopyrum elongatum and wheat has provided further physiological - genetic evidence that GB accumulation contributes to salt tolerance (Colmer et al., 1995). Several groups have taken the first step toward this goal by expressing choline - oxidizing enzymes from bacteria or spinach CMO in tobacco and other plants that do not contain GB (Alia et al., 1998; Nuccio et al., 1998; Sakamoto et al., 1998; Mc Neil et al., 1999). The transgenic plants produced a little GB and in some cases, showed small but significant increase in tolerance to various stresses. The main constraint on GB production in transgenic plants appears to be the endogenous choline supply, because providing choline exogenously leads to a massive increase in GB synthesis (Nuccio et al., 1998). It will therefore most likely be necessary to up- regulate the de nova synthesis of choline in order to increase GB synthesis in non accumulators expressing foreign choline- oxidizing enzymes. Other betaines such as alanine betaine, dimethyl propiothetine and praline betaine have been reported to function as osmoregulators. It has also been shown that 24

levels of aspargines and allantoin increase under salt stress. Sugars and sorbitol also play the role of osmoregulators under salt stress (Loewus and Loewus, 1983; Thomas et al., 1995; Nuccio et al., 1998). Recently diamines like cadaverine, putrescine were shown to increase their amounts in leaf cells and protoplast under osmotic stress or salt stress. (Taraczynski et al., 1993; Nomura et al., 1995; Simon- Sarkadi et al., 2002; Seki et al., 2003).

2.3. Stress on lignification

Lignin is one of the major biomolecule in the biosphere, being second only to cellulose in abundance (Grisebach, 1981 ). Lignification has allowed the evolution of large arborescent land plants capable of survival in relatively arid environments. Because of its functional diversity, its varied distribution in plant tissues, its chemical diversity and also its response towards seasonal changes lignin has been a topic of considerable interest since the early 1950's the mechanism of control of lignin composition and quality have wide implications regarding the adaptations and evolution of land plants and it provide a basis for improved genetic manipulation of lignin. Today a few transgenic plants have been produced with modified lignin. So far, the results obtained with genetically engineered plants with altered lignin have been largely unpredictable. Attempts to alter lignin concentration in plants has focused on the genes encoding the various enzymes with in the monolignol biosynthetic pathway (Hahlbrock, 1976; Hrazdina and Wagner, 1985; Boudet et al., 1995 ; Whetten and Sederoff, 1995; Kato et al., 2000). The last two enzyme of the lignin pathway cinnamoyl CoA reductase (CCR), 25

cinnamyl alcohol NADPH dehydrogenase (CAD) are potential target candidates for genetic manipulation of lignin in that they are solely involved in monolignol biosynthesis. Lignin is a complex hydrophobic net work of phenyl propanoid units results from the oxidative polymerization of one or more of three types of hydroxyl cinnamyl alcohol precursors. These alcohols, para coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol give rise to parahydroxy phenyl lignin, guaiacyl lignin and syringyl lignin respectively. Lignin shows a variety of qualitative and quantitative differences in its distribution. Typically a highly lignified middle lamella containing a predominance of guaiacyl units, and a less lignified secondary wall containing guaiacyl units in gymnosperms, or a mixture of guaiacyl and syringyl lignin units in angiosperms. The type of lignin structural units in angiosperm xylem can often be distinguished on the basis of cell function (structural support versus water conduction - the former favouring a less lignified secondary wall containing syringyl units, the latter favouring a more highly lignified secondary wall containing guaiacyl units). The three mono lignol precursors differ in the extent of methylation a general pathway for lignin biosynthesis has been inferred from studies of specific steps in several diverse species. The mechanisms that control this variation are not well understood. Lignin heterogeneity could be directly related to enzyme diversity and specificity. The initial steps in the biosynthesis of lignin are shared through the general phenyl propanoid metabolism (PPM) and later part of hydroxylation, methylation reactions for the synthesis of mono lignols are carried out by specific enzymes exclusively for lignification. 26

Coniferyl alcohol and other monolignols are derived from phenylalanine in a multistep process (Fig 5). Several other major classes of plant products in addition to lignin are derived from phenylalanine including flavonoids, coumarins, stilbens and benzoic acid derivatives (Dixon and Paiva, 1995; Holton and Cornish, 1995). The initial steps in the biosynthesis of all these compounds are shared through the general phenyl propanoid pathway. The phenyl propanoid compounds are so named because of the basic structure of a three carbon side chain on an aromatic ring, which is derived from L- phenylalanine (Fig. 5). The monolignols themselves are relatively toxic, unstable compounds that do not accumulate in high levels within the living plants. Glycosylation of the phenolic hydroxyl group to produce monolignol glucosides stabilizes the compound and renders them non - toxic. The glucosides probably serve as both storage and transport forms of the monolignols (Whetten and Sederoff, 1995). Enzymes capable of cleaving the glycosidic bonds exist in lignifying cells of many species of plants. The free monolignols are believed to be polymerized to lignin by a free radical mechanism that is initiated through oxidation of monolignols by cell wall bound oxidases.

2.3.1. Enzymes of lignin synthesis

Enzymic reactions of lignin biosynthesis and the mechanism of its control and manipulations for the genetic improvement of plants has recently being reviewed by Grand, (1984); Sederoff et al. (1994); Boudet et al. (1995); Davin and Lewis (2000), Donaldson (2000). At different steps in the 27

o 0- 0 0- 0 0- 0 0- 0 0- 0 0- 0 0- f~ ~'~IZ~I{~,2' ~ 12'7 I{o-'" 0 00 Q~ Y-OH '<0' Y-~ Hoq~ ~~ OH OH OH OH OH lllhenylalanine clnnamale p-eoumarate eaneale !erulale 5 -hydroxyferulate sinapale (6) 14Cl t S-CoA " " 6 OH ~maroyl-eoA ,+'" o H " " 2 Q OH ,tH:oumaraldehyde ~IOAO p-eoumaryl alcohol POD (6) 14Cl o S-eoA " " ~ OH feruloyl-eoA (7) ICCR o H ~ OH con~eraldehyde con~eryl alcohol POD

Lignin

(6) j4Cl o S.CoA.. " ~OO~~ OH slnapoyl-CoA o H " " J~ OH slnapaldehyde slnapyl alcohol Fig. 5 Biochemical pathway of lignin synthesis.

biosynthetic pathway, several enzymes have been identified that could carry out similar reactions during development. Multiple enzymes may also indicate diversity of function or alternate pathway as biosynthesis. 2.3.1.1. Phenylalanine ammonia lyase Phenylalanine ammonia lyase (PAL) is one of the most intensively studied enzymes in plants secondary metabolism because of the key role in phenyl propanoid metabolism (PPM). The deamination of phenylalanine to cinnamate is catalyzed by the enzyme PAL (EC 4.3.1.5). Normally PAL is found as a tetramer in all vascular plants (Czichi and Kindl, 1977; Hanson and Havir, 1981; Jorrin and Dixon, 1990) but an analogous enzyme activity tyrosine ammonia lyase (TAL) that deaminates tyrosine to form para coumarate has been detected mainly in grasses. This activity frequently copurifies with PAL activity, and the question remains whether they are two different proteins or simply two activity of a single polypeptide. Since PAL catalysis the committed step in PPM, it has a regulatory role in controlling the biosynthesis all phenylpropanoid compounds including lignin (Northcote, 1985). Transgenic plants with modified levels of PAL activity have provided opportunities to test hypothesis about the role of PAL in plant metabolism and development (Elkind et aI., 1990; Hao et aI., 1996; Sarma and Sharma, 1999). The amino group cleaved from phenylalanine by PAL is released as ammonia. No data available regarding the fate of this released ammonia, but the potential magnitude of nitrogen loss through this reaction indicates that the ammonia is probably recaptured within the plant. One 28

possible route for recapture of the ammonia is through the action of glutamine synthetase (Lam et aI., 1995). Reductant for incorporation of ammonia into aminoacids can be provided by the oxidative pentose phosphate pathway, even in non photosynthetic plastids (Bowsher et aI., 1992). Bate et al. (1994) analysed phenylpropanoid metabolites in transgenic tobacco plants with decreasing levels of PAL activity and found that lignin content is not greatly affected until PAL activity is reduced to 20 - 25% of wild type levels. Levels of chlorogenic acid and rutin (a flavonoid glycoside) in contrast are affected by small changes of PAL activity. Extensive purification of PAL from cell suspension cultures of bean and alfalfa showed that these species express multiple forms of PAL with different kinetic properties. The individual PAL isozyme display Michaelis-Menten kinetics, but the mixture of isozyme showed the negative cooperativity previously considered characteristic of PAL (Bolwell et aI., 1985; Jorrin and Dixon, 1990). Genes encoding different PAL subunits show tissue specific patterns of expansion in several angiosperms (Bevan et aI., 1989; Lois and Hahlbrock, 1992; EI- Shora, 2002). There has been much speculation about the possibility that different PAL isoforms play different roles in the many aspects of PPM but little experimental evidence is available to test this hypothesis. Recent characterization of PAL genes in Pinus banksiana indicates that four different PAL genes may be present in this species, although the relative levels of activity of the four genes have not been determined (Lam et aI., 1995). PAL isolated from different sources have been discussed in detail by various authors (Hanson and Havir, 1981; Cunha, 1987; Whetten and Sederoff, 1992; Hao et aI., 1996; Sarma and Sharma, 29

1999). More detailed information on the physiological role of this enzyme in lignin biosynthesis and defense mechanism of plant tissues were reported in subsequent papers (cunha, 1987; Goliber, 1989; Morrison and Buxton, 1993; Morrison et aI., 1994; Sewalt et aI., 1997; Jung et aI., 1999). 2.3.1.2. Cinnamyl alcohol -NADPH - dehydrogenase

Cinnamyl alcohol -NADPH- dehydrogenase (CAD EC 1.1.1.195) has been considered to be a marker enzyme for lignification because it occurs after the branch points in PPM for flavonoids and many other phenolic compounds (Walter et aI., 1988; 0' Malley et aI., 1992; Grima­ Pettenati et aI., 1994). The reduction of hydroxy cinnamaldehydes to hydroxy cinnamyl alcohols is catalyzed by CAD. As an indicator of lignin biosynthesis this enzyme has been purified and characterized from Soyabean, Poplar, Spruce, Eucalyptus, and Loblolly pine. Apart from its specific role at the end of lignol biosynthetic pathway CAD also showed its expression in cells that do not make lignin. Previous reports indicated that CAD is expressed in response to stress pathogen elicitors and wounding (Campbell and Ellis, 1992a; Galliano et aI., 1993). Therefore CAD is regulated by both developmental and environmental stimuli eventhough it has a specific role in lignin synthesis. Difference in substrate affinities of CAD enzyme from angiosperms and gymnosperms may playa role in controlling the formation of different types of lignin. Isoforms of CAD with markedly different substrates affinities are detected in species Soyabean, wheat, Eucalyptus and Salix 30

(Wyrambik and Grisebach, 1975; Pillonel et al., 1992; Goffner et al., 1992; Mansell et al., 1976). CAD preparations from gymnosperms are generally much more active on coniferaldehyde, where as angiosperms CAD preparations show more equal activity with coniferaldehyde and sinapaldehyde (Gross, 1985; Mac Kay et al., 1995). The gene encoding CAD has been a target from modification of lignin content in plants through genetic engineering. Tobacco plants transformed with an antisense CAD construct show varying degrees of reduction in CAD activity and modification of phenolic products (Halpin et al., 1992). Modifications of lignin content or quality in forest trees by gene manipulation could improve the processing of wood to make paper. Similarly the reduction of lignin content in forage crops success to improve digestability could have significant application in agriculture. Strategies to modify lignin depend up on the identification of gene that code for or regulate the key steps in PPM and lignin biosynthesis. 2.3.1.3. Peroxidase Two different classes of enzymes peroxidase and laccase have been proposed to perform the polymerization of monolignols in to lignin. The evidence in support of the involvement of these two enzymes is extensive. Laccase (EC 1.10.3.2) an oxygen dependent oxidase contain 4 copper atoms, and peroxidase (POD; EC 1.11.1.7) a hydrogen peroxidase dependent heme protein, both are capable of oxidizing monolignols to free radicals in vitro. Despite 50 years of investigation the exact mechanism and enzymatology of lignin polymerization remain in doubt. The evidence for and

31

against the involvement of laccase and peroxidase in lignification has recently been reviewed (O' Malley et al., 1993; Dean and Eriksson, 1994 ). A convincing answer to this question will require demonstration of changes in lignin content or composition upon experimental manipulation of enzyme activity, either in transgenic plants or in mutants are under way in several laboratories. Apart from the role of POD in the last step of lignification POD has been implicated in numerous physiological processes including cross linking of cell wall polysaccharides, pathogen resistance, oxidation of fatty acid and phenols, phytohormone catabolism, fruit ripening (Smith, 1982; Lee et al., 1984; Fry, 1980; 1986; 1987; Marin and Cano, 1992; Stinzi, 1993; Polle, 1994; Alcazar, 1995; Civello, 1995; Brett and Waldron, 1996; Florence and Frederic, 1997; Brett, 1999; Gonzalez et al., 2000; Salini and Mohankumar, 2001 ). POD activity has been used as an index of various operations associated with the processing of vegetables and fruits due to its stability. This enzyme has attracted in the food industry because its ability to bring out desirable and sometimes undesirable changes. POD is well suited for the preparation of the enzyme conjugated antibodies due to its ability to yield chromogenic products at low concentration and is relatively good stability characteristics. It has a greater potential for application in the area of waste water treatment once activated by hydrogen peroxide, it catalyzes the oxidation of variety of toxic aromatic compounds including phenols, biphenols, anilines, benzidines and related heteroaromatic compounds (Karam and Nicell, 1997). During the peroxidatic reduction of the enzyme an electron 32

donor is an essential factor for the splitting of one molecule of hydrogen peroxide to two molecules of water. Based on the nature of the electron donor the hydrogen peroxide scavenging peroxide can be classified in to ascorbat peroxidase, guaicol peroxidase and cytochrome peroxidase. However the molecular property of ascorbate peroxidase is very different from those of guaicol peroxidase, in addition to their difference in specificities for the electron donor (Asada, 1992b; Takahama and Oniki, 1997). The most conspicuous difference between the two types of POD's are their amino acids sequence Le., ascorbate peroxidase show a high degree of homology to those of cytochrome C peroxidase (from yeast) rather than those of guaicol peroxidase from plants.

2.4. Oxidative stress

The common· stress factors that induce the growth of mangroves in the varied habitat viz. tide dominated, basin and riverine are anoxygenic condition, osmotic factors and gravitational stress (Allen et ai., 1995; Bohnert and Sheveleva, 1998; Knight and Knight, 2001; Taylor et aI., 2002; Blokhina et ai., 2003). Histomorphologically this ecological group expressed its identity by the presence of structures like pneumatophores, knee roots, succulent leaves, sunken stomata and colorless tissue in the mesophyll. Since the histological adaptations directly or indirectly reflect the physiology of the plant, the cellular functions will also get changed. The accumulation of dioxygen in earth's atmosphere allowed for the evolution of aerobic organisms that use O2 as the terminal electron acceptor, thus

33

providing a higher yield of energy. Thus O2 which is essential for the existence and survival of aerobic life presents living organisms with a variety of physiological challenges called collectively as 'oxidative stress' (Arora et aI., 2002). These challenges may be greater for plants because of their stationary life style under constantly changing environments and also because plants consume O2 during respiration and generate it during photosynthesis. Oxidative stress is induced by a wide array of environmental factors in which O2 deprivation stress is the most significant in the case of mangroves. This physiological stress of O2 shortage is distinguished by three different physiological states: (i) transient hypoxia (ii) anoxia (iii) reoxygenation.

2.4.1. Reactive oxygen species

The generation of reactive oxygen species (ROS) is characteristic for hypoxia and especially for reoxygenation (Crawford and Braendle, 1996; Biemelt et aI., 1998 Rio et aI., 1998; Ramonell et aI., 2001). Generation of ROS in plants has been implicated in biotic and abiotic stress. In the ground state the molecular oxygen is relatively unreactive, yet it is capable of giving rise to lethal reactive excited states as free radicals and derivatives (Smirnoff and Cumbes, 1989; Foyer et aI., 1994). Free radical can be defined as chemical species possessing an unpaired electron, which is formed by homolytic cleavage of a covalent bond of a molecule, by the loss of a single electron, from a normal molecule or by the addition of a single electron to a normal molecule (Ray and Husain, 2002). All of these activated O2 species are extremely reactive and cytotoxic in all organisms. These highly 34

reactive species can react with unsaturated fatty acids to cause peroxidation of essential membrane lipids in the plasma membrane or intracellular organelles. The possible examples are (i) Super oxide anions (02'-); (ii) Hydroperoxyl radical (H02); (iii) Peroxide ion (H02); (iv) Hydrogen Peroxide (H202); (v) Hydroxyl radical (.OH). They are otherwise called as reactive oxygen metabolites (ROMs) (Green and Hill; 1984, Chessman and Slater 1993). 2.4.1.1. Super oxide anion (02' -) Super oxide anion is the first reduction product of O2 , O2 + e- ~ O2'-. It is a base with the equilibrium with its conjugate acid, the hydroperoxyl radical H02', O2'- is a relatively non-reactive species and dismutates to H20 2 . The reaction either occurs spontaneously or is catalyzed by the enzyme superoxide dismutase (SOD). There are two sites of O2'- production on the reducing site of photosystem I (Badger, 1985; Gupta et ai., 1993b; Asada, 1994). The majority of O2 reduction in vivo is thought to proceed via reduced ferredoxin (Fd red) which reduces molecular oxygen to the O2'-, (Reaction 1), H20 2 is then formed through dismutation of O2'-. The latter occurs spontaneously (Reaction 2 ) or by SOD (Reaction 3). (Reaction 1) (Reaction 2) 202'- + 2H+ SOD) H20 2+ 02 (Reaction 3) O2'- directly affect some intercellular enzymes, chromosome breakage and peroxidation of unsaturated lipids. 35

2.4.1.2. Hydrogen peroxide (H202)

It is the most stable ROS. It is the least reactive and the one that can be readily detected (Gillham and Dodge, 1986). H202 may be generated directly by divalent reduction of 02 (Reaction 4) or indirectly by a univalent reduction of 02·- (Reaction 2 or Reaction 3). H202 is very sensitive to decomposition and it is catalysed by catalase (CAT) and Peroxidase (POD). 02 2H + H202 2H202 �2H20+02 (Reaction 4) 2 H2 02 � 2H20 + 02 H202 in presence of metal ions forms .OH Fe 2 + + H202 � Fe3+ + · OH + OH- (Fenton reaction) H202 can also reacts with 02·- to form .OH in presence of iron (Haber Weiss reaction). H O O Fe 2+ H OH 2 2 + 2 02 + .0 + - H202 is the more essential species to induce cell injury (Groden and Beck, 1979; lnze and Camp, 1995; Gechev et al., 2002). 2.4.1.3. Hydroxyl radical (' OH)

Hydroxyl radical (' OH) is highly reactive. It can react with any molecule present in cells and is therefore short lived. H202 + 02 ·- can form · OH that can initiate lipid peroxidation, attack DNA proteins and many small molecules. ·OH species are formed either. or 36

Metal ion Cu+, Cu2+ can replace Fe2+, Fe3+ in these reactions. Oxidation of organic substrates may proceed by two possible reactions: (i) addition of· OH to an organic molecule, or (ii) abstraction of a hydrogen atom from it. In the former reaction the· OH add to organic substrate forming a hydroxylated product, which is further oxidized by Fe3+, O2 or other agents to a stable oxidized product. The hydroxylated product can also dismutate to form cross linked products (Pryor, 1986). R + .OH -). ROH ROH + Fe3+ -). ROH + Fe 2+ ROH+0 2 -).ROH+0 2 ROH+ROH+2H+ -). R-R+2H 20 In the second reaction, the .OH radical oxidizes the organic substrate by forming H20 and an organic radical. The latter product has single unpaired electron and thus can react with oxygen in triplet ground state. The addition of triplet oxygen to the organic radical can lead to the formation of a peroxy-radical, which can readily abstract hydrogen from another organic molecule leading to the formation of a second organic radical. This chain reaction is far more damaging than any other reaction catalysed by ROS. .R + O2 -? .ROO .ROO + RH ~ .R + ROOH Peroxidation damage of plasma membrane leads to leakage of cellular contents, rapid desiccation and cell death. Intracellular membrane damage can affect respiratory activity in mitochondria, cause pigment 37

1f."'..'.'.. '9Ja.Wt-~" ..'/.. /" \' breakdown and cause loss of carbon fixing ability in chloroplast (Scandalios, 1993; Oat et aL, 2000; Mittler, 2002). Several Calvin cycle enzymes within chloroplast are extremely sensitive to H202 and high levels of H20 2 as the product of superoxide dismutation directly inhibit CO2 fixation (Kaiser, 1979; Gogorcena et aI., 1995). H20 2 has also been shown active with mixed function oxidases in marking several types of enzymes for proteolytic degradation (Fucci et aI., 1983). In plants, the superoxide radical and singlet oxygen are commonly produced in illuminated chloroplast by the occasional transfer of an electron from an excited chlorophyll molecule or PS I components under conditions of high NADPH/NADP ratios to molecular oxygen (Knox and Dodge, 1985). In addition to normal metabolic activity ROS can result from cellular exposure to various environmental stimuli such as UV light and other forms of radiation, herbicides, pathogens, certain injuries, hyperoxia, ozone, temperature fluctuations and various other stresses in most aerobic organisms (Scandalios, 1992; Gogorcena et aI., 1995). ROS are produced in significant quantities in various subcellular compartments I organelles. Each organelle has a potential target for oxidative stress as well as mechanism for eliminating the noxious oxyradicals (Table 3). 2.4.2. Antioxidant and scavenging enzymes

Oxygen is essential for aerobic life processes. However cells under aerobic condition are threatened with result of ROS that are efficiently 38

Sub Cellular Type of active 02

Location species

Chloroplast Superoxide, H202 Mitochondria Superoxide, H202 Cytosol Superoxide, H202 Glyoxysomes and H202 Peroxisomes

Source of active 02 Species

PS II, Enzymic Electron transport and enzymic Enzymic � oxidation Photorespiraton

Enzymic Scavenging

Systems

SOD, Ascorbate peroxidase SOD, Peroxidase, CAT SOD, CAT Peroxidase CAT

Table 3. Potential targets for oxidative stress in cell system.

taken care of by the powerful antioxidant systems (Ray and Husain, 2002). Aerobic life is characterized as continuous production of oxidants balanced by equivalent synthesis of antioxidants (Rice-Evans and Diplock, 1993). The improper balance between ROS production and antioxidant defenses result in 'oxidative stress' which deregulates the cellular function leading to various pathological conditions (Bandyopadhyay and Banerjee, 1999; Taylor et aI., 2002). The elimination of free radical and ROS becomes a target for antioxidant enzyme system for normal metabolic processes. The first line of defense against ROS mediated cell damage are antioxidant enzymes - super oxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT). The term antioxidant has been defined as any substance that delays or inhibits oxidative damage to a target molecule (Halliwell and Gutteridge 1999; Noctor et aI., 2002). 2.4.2.1. Superoxide dismutase (SOD; EC: 1.15.1.1) The biological role and significance of SOD as a protective enzyme against O2 toxicity are born out in numerous studies with prokaryotes, lower and higher eukaryotes including higher plants. The role of SOD has been extensively revealed recently (Fridovich, 1986; Hassan and Scandalios, 1990; Scandalios 1990, 1992; Bowler et aI., 1992; Gralla and Kosman, 1992; Gupta, 1993b; Ray and Husain, 2002; Blokhina et aI., 2003). SOD was first isolated from bovine blood as a green copper protein whose biological function was believed to be copper storage (Mann and Keilin, 1938). Over the years the enzyme has been variably referred to as indophenol oxidase and 39

tetrazolium oxidase. The catalytic function of the enzyme was discovered by McCord and Fridovich (1969). The enzyme is ubiquitous, being widely distributed among O2 consuming organisms, aerotolerant anaerobes and some obligate anaerobes· (Fridovich, 1986; Cross and Jones, 1991). Structurally SODs, are multimeric metalloprotein that are very efficient at scavenging the superoxide radicals. Three distinct types of SODs based on the metal ion in their active sites have been observed from a wide range of organisms examined. Thus there are SODs that contain copper, zinc and manganese or iron (Duke and Salin, 1985). SODs are generally formed in cytosol, chloroplast, matrix of mitochondria, but no extracellular SODs have been reported in plants (Marklund, 1984). At present the evolutionary origin of these 3 classes of SOD proteins are not clear however the sequence data suggest that three types of SOD fall in to two phylogenetic families i.e. Cu/Zn SOD and Fe/Mn 30 D. The available data suggest that the two families of SOD must have evolved independently and they are selected in response to a common environmental stress, the oxygenation of the biosphere by photosynthetic organisms. The SOD enzyme converts the potentially dangerous O2' - to H20 2. Thus averting cellular damages.

2.4.2.2. Catalase (CAT; E.C:1.1.1.6)

Catalase as a combined action converts the released H20 2 by SODs activity to H20 and molecular oxygen. This enzyme is present in most cells. As an enzyme indispensable for stress defense in C3 plants its primary function is to protect cells against H202. Plant cells are relatively tolerant to 40

exogenous HzOz in comparison with the mammalian cells and this is generally thought to be due to the presence of extra cellular peroxidase in the plant cell wall (Gregory, 1968; Kellog and Fridovich, 1975; Dhindsa et aI., 1982; Streb et aI., 1993; Levine et aI., 1994; Guyton et aI., 1996 Willekens et aI., 1~97; Yang and Poovaiah, 2002; Pons and Welschen, 2003). Catalase comprises 10-25% of total peroxisome. proteins and its extreme activity in peroxisome due to its abundance in the organelle. The enzyme has been purified from leaves, green cotyledons and yeast. The enzyme consists of 4 identical subunits with heme as its prosthetic group and has a molecular weight of 225kD to 300 kD. In the exhaustive review of Deisseroth and Dounce (1970), the catalase activity on scavenging HzOz radical was interpreted in two ways. (ii) Catalase. HzOz complex + HzOz~ Catalase + 2HzO+Oz (iii) Catalase. HzOz complex + HzR ~ Catalase + 2HzO+R The reaction (i) and (ii), the catalase action is considered as the catalatic reaction of the enzyme. If the availability of HzOz in the cell system is not enough for a catalatic action, the activity of the enzyme is triggered in a peroxidative way. In other words the level of HzOz in the cell-system becomes a decisive factor of the catalase activity (Huang et aI., 1983). Catalase activity in isolated peroxisomes can be demonstrated with the electron microscope by incubating cells in 3,3'diamine benzidine (DAB) and HzOz to cytochemically localize catalase reactivity. Osmium black formed at the site of enzyme

41

reactivity in a high resolution electron dense stain, which is easily recognized in sections. Thus fine structural descriptions are often accompanied by DAB cytochemistry to define the distribution and location of peroxisomes among and within cells (Vigil, 1970; 1973; del Rio, 1996; Talarczyk et al., 2002).

2.4.2.3. Glutathione peroxidase (GPx; EC: 1.11.1.9)

Glutathione peroxidase is a well known antioxidant enzyme for scavenging ROSs, which inturn requires glutathione as cofactor. Among the many functions of glutathione, it is involved in the generation of the nucleotide precursors of DNA via, the reduction of ribonucleotides to deoxy ribonucleotides (Meister, 1991; Foyer et al., 1997; Roxas et al., 2000). Glutathione peroxidase (GPx) catalyses the oxidation of Glutathione (GSH) to Glutathione disulphate (GSSG) at the expense of H202. By its selenium dependency Gpx can be divided into two forms: Selenium dependant GPx and Selenium independent GPx. The former is a tetramer of molecular weight 84 kD with very high activity towards both H202 and organic hydro peroxide. This enzyme is found in both cytosol (20%) and mitochondria (30%) of various tissues. lodoacetate and cyanide are considered as the inhibitor of the enzyme (Blum and Fredovish, 1985).

2.5. C2 pathway in plants

Photorespiration is of great interest because of its relationship to plant productivity. It has been estimated that in C3 plants, the process of photorespiration oxidizes up to 50% of the synthesized photosynthetic 42

products, where as in C4 plants photorespiration is either not present or occurs in much small amounts (Grodzinski, 1978; Somerville and Ogren, 1981; 1982; Sharkey, 1988; Lidon and Henriques, 1993; Wingler et al., 1999, 2000). Control of photorespiration has emerged as a primary objective in efforts to increase plant productivity in those crops possessing the C3 pathway of photosynthesis. Recent progress in characterizing photorespiration and its regulation has identified the target sites for modifying this process and provided explicit approaches which in principle can create and select for plant strains with reduced photorespiration rate (Ogran, 1984 ). The earliest report of manifestation of photorespiratory activity was made by Warburg, who observed 02 inhibited photosynthesis in Chlorella (Warburg, 1920). A second manifestation of photorespiration, 02 stimuated CO2 evolution in the light was found by Decker (1955). Additional important advances in understanding photorespiration were made by several workers (Warburg and Krippahl, 1960; Krotkov, 1963; Forrester et al., 1966; Bowes and Berry, 1972; Kennedy, 1976; Kaiser, 1979; Tolbert, 1980; Heupel and Heldt, 1994; Sharkey , 1988; Raghavendra et al., 1998; Ramonell et al., 2001; Haupt- Herting and Fack, 2002; Ueno et al., 2003). Photosynthetic glycolate formation is a complex irreversible process, which at least in intact system cannot be uncoupled from photosynthetic carbon metabolism. Available information concerning glycolate synthesis is a feature of several aspects of plant carbon metabolism attracted the attention of plant physiologists (Tolbert, 1971; 1979; 1980; Tolbert and

43

Essner, 1981; Zelitch, 1972b; 1975; Chang and Hunang, 1981; Lorimer, 1981; Husic et al., 1987; Dai et al., 1993; Raghavendra et al., 1998; Rio La- del et al., 2002). Carbon flow in. to the photosynthetic carbon cycle or in to the photorespiratory process of glycolate biosynthesis and metabolism is determined by the dual function of ribulose 1, 5 - biphosphate carboxylase I oxygenase. The competition between a continuum of changing CO2 and 02 concentrations for these initial reactions is a major factor that produces different rates of photosynthesis and photorespiration (Bowes, 1991; Lidon and Henriques, 1993; Douce and Neuberger, 1999). The fixation of CO2 yields 3 - phospho glycerate, most of which is reduced to carbohydrates. Fixation of 02 yields a two carbon phospho glycolate and 3- phosphor glycerate. Hence this pathway is also called as C2 pathway (Hu sic et al., 1987; Noctor et al., 2002). The phospho glycolate is hydrolysed by a specific chloroplastic phosphatase and the glycolate is excreted (Richardson and Tolbert, 1961 ). Metabolism of glycolate carbon occurs sequentially in three organelles, the peroxisomes, the mitochondrion and the chloroplast. The initial substrate for photorespiration, phospho glycolate is produced in the chloroplast. Leaf peroxisomes, a specialized microbody, play a role in the irreversible portion of photorespiration (Tolbert, 1980; Heupel and Heldet, 1994 ). They are involved in the formation of glycerate from glycolate. Glycolate oxidase, the key enzyme involved in the oxidation of glycolate to glyoxylate and hydrogen peroxidase (H202). Although the enzyme is present 44

in the peroxisomes of non green tissues its specific activity in leaf peroxisomes is more than ten times higher than that in the isolated peroxisomes of other plant and animal tissues (Huang et al., 1983). Such a high activity signifies the importance of this enzyme in the leaf peroxisomes. The enzyme activity per gram fresh weight of leaves is high enough to a account for all the metabolism of glycolate during active photorespiration (Zelitch, 1972a). The released H202 was decomposed by the catalatic action (Deisseroth and Dounce, 1970). The glyoxylate released by the oxidation of glycolate was transaminated to glycine by either glutamate or serine (Kisaki et al., 1971 ). There are two different aminotransferase; one for glutamate: glyoxylate and other for serine: glyoxylate. The glyoxylate is converted in to glycine via the transamination reaction. The glycine formed is transported out of peroxisomes in to mitochondrion where two molecules of glycine react to produce serine, CO2 and ammonia. This process consists of several reactions involving tetrahydrofolate (C1 -THFA) bound to a mitochondrial enzyme system. Oxidative glycine decarboxylation is linked to NAO reduction and yields CO2 from the carboxyl group of glycine, ammonia and C1 - THFA from C2 of glycine (Wu et al., 1991; Dutilleul et al., 2003). The CO2 may be either refixed photosynthetically, or it may escape from the leaf as an indication of photorespiration. Serine produced is transported out of mitochondria and in to peroxisomes where it is converted successively to hydroxypyruvate and glyceric acid. Finally, glyceric acid is transported out of peroxisomes in to

45

chloroplasts, where it is phosphorylated by ATP to 3 - phospho glyceric acid pool of the photosynthetic carbon reduction cycle (Rathnam and Chollet, 1979; Upadhye and Karadge, 1991; Leegood et al., 1995). The occurrence of photorespiration is an enigma and serves to dissipate excess photochemical energy (Foyer and Hall, 1980; Jordan and Ogren, 1983; Ogren, 1984 ). Photorespiration is essentially nonproductive photosynthetically because the carbon is used to regenerate ribulose biphosphate (RuBP), with no gain in carbohydrate synthesis for respiration or storage. At relatively high light intensities, photosynthesis in C3 plants is said to be saturated because the rate of photorespiration is equal to net rate of CO2 fixation at higher intensities, photorespiration predominates, with an appreciable loss in CO2 fixation. It is interesting to note that the CO2 release from photorespiring cells is a reflection of amount of CO2 not fixed due to 02 fixation. The actual CO2 product of photorespiration, however, is the CO2 released in the production of serine from two glycines. Paradoxically, the high light intensities are effective in ATP and NADPH synthesis through photophosphorylation and reduction. The ATP and NADPH are required to regenerate the RuBP for photorespiration as well as CO2 fixation. The serine produced may be incorporated in to protein or converted to carbohydrates via its direct conversion to 3 - phosphoglyceric acid. Actually 3- phosphoglyceric acid may be produced from 3 carbon acids as well as from glycolate acid, glyoxylic acid, glycine and the serine. The

46

formation of 3 - posphoglyceric acid from these latter compounds is referred to as the glyolate pathway. Some researchers speculate that photorespiration evolved as a pathway in response to phosphoglycolate accumulation and a regulating mechanism for the levels of phosphorylated sugars. It may also be important as a mechanism for the intra cellular transport and inter conversion of carbohydrates and nitrogenous compounds (glycolate to glycine to serine to phosphoglyceric acid) (Herber and Krause, 1980; Kiirats et al., 2002; Tezera et al., 2003) . 47

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