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International Journal of Research in Plant Science

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ISSN 2249-9717 Original Article Effect of NaCl Stress on Biochemical and Enzymes Changes of the Halophyte Suaeda maritima Dum.
M. Rajaravindran and S. Natarajan* Department of Botany, Annamalai university, Annamalainagar, Tamilnadu, India- 608 002 Email; s.natarajan20@yahoo.com Received 01 February 2012; accepted 15 February 2012 Abstract The present study was made to study the effect of different concentrations of sodium chloride on biochemical constituents and antioxidant enzymes of the seedlings of Suaeda maritima. The plant could survive a wide range of 100-500 mM NaCl concentrations. The upper limit for the survival of the seedlings was 300 mM NaCl. Organic compounds such as amino acids and sugar decreased upto optimum level of 300mM NaCl concentration. The starch and chlorophyll content increased upto 300mM NaCl. Beyond these levels the content decreased marginally. The proline content increased with the increasing concentration of the salt. The antioxidant enzymes such as catalase (CAT), peroxidase (POX) and poly phenol oxidase (PPO) increased up to the optimum level of 300mM NaCl concentration and beyond these levels the contents decreased marginally. 2011 Universal Research Publications. All rights reserved Key words: NaCl, Salinity,Halophytes,Organic constituents, antioxidant enzymes, Suaeda maritima INTRODUCTION Salinity stress is one of the most significant limiting factors in agricultural crop productivity (Boyer, 1982). Salinity and drought are among the major stresses that adversely affect plant growth and crop productivity. These constraints remain the primary causes of crop losses worldwide, reducing average yields by more than 50% (Boyer, 1982; Wang et al., 2003). Salt stress alters various biochemical and physiological responses in plants, and thus affects almost all plant processes including photosynthesis, growth and development (Iqbal et al., 2006). Salt induces osmotic stress by limiting absorption of water from soil, and ionic stress resulting from high concentrations of potentially toxic salt ions within plant cells. Plants have evolved a variety of protective mechanisms to allow with these unfavorable environmental conditions for survival and growth including the accumulation of ions and osmolytes such as proline. The accumulation of these compounds prevents water loss and ion toxicity. The alleviation of oxidative damage and increased resistance to salinity and other environmental stresses are often correlated with an efficient antioxidative system (Jaleel et al., 2007 a,b; Manivannan et al., 2007). Salinization of agricultural soils is a worldwide concern, especially in irrigated lards. Saline soil is characterized by the presence of toxic levels of sodium and its chlorides and sulphates. Salt tolerance is the ability of plants to grow and complete their life cycle on a substrate that contains high concentrations of soluble salt. Plants that can survive on high concentrations of salt in the rhizosphere and grow well are called halophytes. Salinity tolerance is defined as the ability of plants to continuously grow under salt stress conditions (Munns, 2002).iAnother major factor of salt tolerance mechanisms is the ability of plant cells to adjust osmotically and to accumulate organic solutes (proteins, sugar, amino acids, etc.). The accumulation of these compounds is not only important for cell osmoregulation but also for the protection of subcellular structure (Munns, 2002) and maintenance of protein structures. Many of the physiological adaptations of plants to saline conditions are similar to the adaptations shown by plants to desiccation stress and it has been suggested that plants showing drought resistance would also

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exhibit salinity tolerance (Munns, 2002). However, in some halophytes, the salt tolerance mechanisms are not sufficient for tolerance of drought or frost (Ueda et al., 2003). Halophytes and other salt-tolerant plants may provide sensible alternatives for many developing countries (Squires and Ayoub, 1994). MATERIALS AND METHODS Plant Materials and Treatments Healthy seedlings of Suaeda maritima were up rooted without damaging the root system and brought to the laboratory in polythene bags. Uniform sized and healthy seedlings after a through wash in the tap water were planted individually in polythene bags (7 5). The polythene bags filled with homogenous mixture of garden soil containing red earth, sand and farmyard manure (1:2:1). The seedlings were irrigated with tap water and allowed to establish well. After establishment in polythene bags for 30 days about 300 healthy plants were subjected to saline treatment. The treatment constituted control, 100, 200, 300, 400 and 500 mM NaCl concentration. At higher concentrations, seedlings mortality occurred within a week after the salt treatment. The experimental plants thereafter, maintained upto 500mM NaCl. Salt solutions were prepared with NaCl (Laboratory Grade, Glaxo Laboratories, India). The treatments were continued until the plants received the required concentrations of the salt, after this all the plants were irrigated with tap water. The experimental yard was roofed with transparent polythene sheet at the height of 3 m from the ground in order to protect the plants from rain. Sampling for various studies was taken on the 60th day after NaCl treatment. Estimation of total free amino acids Total free amino acid content was estimated by the method Moore and Stein (1948), the leaf, stem and root tissues were treated with 80 per cent boiling ethanol for taking extracts (5 ml extract representing 1 g tissue). One ml of ethanol extract was taken in 25 ml test tube and neutralized with 0.1 N sodium hydroxide using methyl red indicator. One ml of ninhydrin reagent was added (800 mg stannous chloride in 500 ml citrate buffer pH, 5.0, 20 g ninhydrin in 500 ml methylcellosolve, both solutions were mixed). The contents were boiled in a water bath for 20 minutes and 5 ml of diluting solution (distilled water and n-propanol mixed in equal volume) was added, cooled and diluted to 25 ml with distilled water. The absorbance was measured at 570 nm in a spectrophotometer (U-2001, HITACHI). Estimation of total sugars Total sugars were determined by Nelson (1944), One ml of ethanol extract taken in the test tube was evaporated in a water bath. To the residue, 1 ml of distilled water and 1 ml of 1 N sulphuric acid were added and incubated at 49C for 30 minutes. The solution was neutralized with 1N sodium hydroxide using methyl red indicator. One ml of Nelson reagent was added to each test tube prepared by mixing

reagent A and B in 25:1 ratio (Reagent-A: 25 g sodium carbonate, 25 g sodium potassium tartarate, 20 g sodium bicarbonate and 200 g anhydrous sodium sulphate in 1000 ml; Reagent B: 15 g cupric sulphate in 100 ml of distilled water with 2 drops of concentrated sulphuric acid). The test tube were heated for 20 minutes in a boiling water bath, cooled and add 1 ml of arsenomolybdate reagent (25 g ammonium molybdate, 21 ml concentrated sulphuric acid, 5 g sodium arsenate dissolved in 475 ml of distilled water and incubated at 37C in a water bath for 48 h) was added. The solution was thoroughly mixed and diluted to 25 ml and read at 495 nm in a spectrophotometer. The reducing sugar contents of unknown samples were calculated from glucose standards. Estimation of protein Protein was assayed as described by Lowry et al. (1951), to 0.5 ml of protein extract, 5 ml of the reagent C was added (prepared by mixing reagent A and reagent B in 25:1 ratio; Reagent-A: 400 mg of NaOH was dissolved in distilled water and made upto 100 ml. To this solution, 2 g of Na2CO3 was added. Reagent-B: 2 g of CuSO4 was dissolved in distilled water and made upto 100 ml and 2 g sodium potassium tartarate was dissolved in distilled water and made upto 100 ml, both solution were mixed equal volume) and it was allowed to stand for 10 minutes at 28C. 0.5 ml of folin-phenol reagent (Folin-ciocalteu and distilled water were mixed in the ratio 1:2 (v/v)) was added to this solution and kept at room temperature (30C) for 10 minutes and the absorbance was read at 660 nm in a spectrophotometer. The protein contents of unknown samples were calculated from Bovine Serum Albumin standards. Estimation of proline Proline was assayed according to the method Bates et al. (1973). Five hundred mg of plant tissue was homogenized in 10 ml of 3 per cent aqueous sulphosalicylic acid. The homogenate was filtered through Whatmann No. 42 filter paper. Two ml of acid ninhydrin (1.25 g ninhydrin in 30 ml of glacial acetic acid and 20 ml of 6 M phosphoric acid) and 2 ml of glacial acetic acid in a test tube was heated for an hour at 100C. The reaction mixture was extracted with 4 ml of toluene and mixed vigorously by using a vortex mixture for 15-20 seconds. The chromophore containing toluene was aspirated from the aqueous phase. The absorbance of the toluene layer was measured in a spectrophotometer at 520 nm using toluene as blank. Estimation of chlorophyll Chlorophyll content was estimated by the standard method Arnon (1949). Five hundred mg of leaf tissue was taken in a pestle and mortar with 10 ml of 80 per cent acetone and it was ground well. Then the homogenate was centrifuged at 800 g for 10 minutes and the supernatant was saved. The pellet was re-extracted with 5 ml of 80 per cent acetone each time till the pellet become colorless.

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Estimtaion of catalase (EC 1.1.1.6) Catalase activity was assayed as described Chandlee and Scandalios (1984). 500 mg of frozen material was homogenized in 5ml of ice cold 50 mM PMSF (Phenyl methyl sulfonyl fluoride). The extract was centrifuged at 4C for 20 min at 12500 rpm. The supernatant was used for enzyme assay. The activity of enzyme catalase was measured using the method of Chandlee et al. (1984) with modification. The assay mixture contained 2.6 ml of 50 mM potassium phosphate buffer (pH 7.0), 0.4 ml of 15 mM H2O2 and 0.04 ml of enzyme extract. The decomposition of H2O2 was followed by the decline absorbance at 240 nm. The enzyme activity was expressed in units per min per mg protein. Estimation of peroxidase (donor: hydrogen peroxide oxidoreductase; (EC. 1.11.17) Peroxidase activity was assayed by the method Kumar and Khan (1982). Assay mixture of peroxidase contained 2ml of 0.1 M phosphate buffer (pH 6.8), 1ml of 0.001M pyrogallol, and 1ml of 0.005M hydrogen peroxide and 0.5ml of enzyme extract. The reaction mixture was incubated for 5 minutes at 25C, after which the reaction was terminated by adding 1ml of 2.5 N sulphuric acids. The amount of purpurogallin formed was determined by reading the absorbance at 420 nm against a blank prepared by adding the extract after the addition of 2.5 N sulphuric acid at zero time. The activity was expressed in unit per minute per mg protein. Estimation of polyphenoloxidase (O-Diphenol: O2 oxidoreductase, EC. 1.10.3.1) Polyphenol oxidase activity was assayed Kumar and Khan (1982). Assay mixture for polyphenol oxidase contained 2 ml of 0.1M phosphate buffer pH (6.0), 1ml of 0.1M catechol and 0.5 ml of enzyme extract. This was incubated for 5 minutes at 25C, after which the reaction was stopped by adding 1ml of 2.5N sulphuric acid. The absorbance of the purpurogallin formed was recorded at 495 nm. The enzyme activity was expressed in units. One unit is defined as the amount of purpurogallin formed, which raised the absorbance by 0.1 per minute under the assay condition. Statistical analysis Each treatment was analyzed with at least five replicates and a standard deviation (SD) was calculated; data are expressed in mean SD of five replicates. RESULTS AND DISCUSSION Effect of salinity on amino acid content

content. Greater accumulation of amino acids was also observed in halophytic plants of Aeluropus logopoides and Sporobolus madraspatanus in response to increased seawater salinity in growth medium (Joshi and Misra, 2000). Similar observations were made in other halophytes such as Helochola setulasa (Joshi et al., 2002). A gradual increase in amino acid at high salinity level could be due to increased degradation of protein. Proline was the most abundant amino acid that increased with NaCl treatment in the plants.

Fig.1.Effect of NaCl on amino acid content (mg g-1 fr. Wt.) of Suaeda Maritima on 60th day qfter treatment.

Effect of salinity on protein content

Fig.2.Effect of NaCl on total sugar content (mg g-1 fr. wt.) of Suaeda maritima on 60th day after treatment.

Changes in the protein content in the leaf stem and root in response to different concentrations of NaCl are given in (Fig. 2). The protein content increased with increasing NaCl salinity upto 300 mM in Suaeda maritima. And at higher concentrations it steadily declined. The changes in soluble protein showed a reverse trend to that of free amino acids implying that the increase in protein content may be at the The results on the effect of NaCl salinity on the free expense of amino acids and that the salinity changes amino acid are presented (Fig. 1). The amino acid content of influenced the inter conversion of these compounds. In the leaf, stem and root decreased with increasing NaCl halophytes, the protein content increased with increasing concentrations upto 300 mM in Suaeda maritima. Of the concentrations such as Helochola setulosa (Joshi et al., three tissues the leaf had more amino acids then the other two 2002), and Thellanjiella halophila (Mrah et al., 2006). In tissues. The total free amino acid content was found to general, the protein content increased with increasing decrease gradually with increasing concentration of NaCl concentration upto an optimal level. Beyond the optimum treatment upto 300 mM respectively, and at higher level, the protein content decreased in Sesuvium portulacastrum concentrations, there was a gradual increase in the amino acid (Venkatesalu et al., 1994) and Helianthus annuus (Manivannan International Journal of Research in Plant Science 2012; 2(1): 1-7

et al., 2008). Protein content in the tissues of many plants declined under drought or salinity stress, because of proteolysis and decreased protein synthesis (Joshi and Misra, 2000). Effect of salinity on sugar content Changes in total sugar content in leaf stem and root in response to different concentrations of NaCl are given in (Fig.3). The total sugar content decreased with increasing concentrations upto 300mM NaCl. At higher concentrations, total sugar content increased gradually. The total sugar content had decreased with increasing NaCl salinity up to the optimum level and at higher salinities the sugar content had increased. The higher sugar content in the extreme salinity levels is regarded as an additional mechanism to prevent salt injury (Rathert, 1982). Under severe salinity stress, a low carbohydrate level could be due to either high respiration or a decrease in photosynthetic activity accompanied by reduction in growth rate. The changes in the carbohydrate are regulation primarily by K+ and Cl- ions (Rathert, 1982). An increasing sugar content and corresponding decrease in the starch at higher salinities have been reported in several halophytes (Joshi et al., 2002; Ashraf and Haris, 2004). Sugars are the source of energy and carbons needed for adaptative or defensive responses to stresses.

Fig.4. Effect of NaCl on proline content (mg g-1 fr. wt.) of Suaeda maritima on 60th day after treatment stress induces proline accumulation in many halophytes (Brown and Pezeshki, 2007; Song et al., 2006).The present observations are in accordance with several studies that proline content progressively increased with high levels of salinity in Thellungiella halophilla (Inan et al., 2004) and Sesuvium portulacastrum (Ramani et al., 2006). Proline may act as an enzyme protectant stabilizing the structure of macromolecules and organelles. Recent studies indicate that adaptation to salinity is closely associated with proline accumulation. A significant increase in proline content was found only at high salinity salinity (Wang, 2006). Effect of Salinity on chlorophyll content

Fig.3.Effect of NaCl on protein content (mg g-1 fr. wt.) of Suaeda maritima on 60th day after treatment. An increasing trend in chlorophyll content of the leaf was noticed (Fig.5) with increasing NaCl concentration upto Effect of salinity on proline content The effect of NaCl on the proline content in the leaf, 300mM NaCl on the 60th day of salt treatment and thereafter, stem and root are given in (Fig. 4). There was a gradual rise it steadily declined. The chlorophyll a was always higher in the level of proline in all the three tissues on 60th day than that of chlorophyll b at all concentrations. The sampling with increasing concentration of NaCl. The leaf chlorophyll content of Suaeda maritima was found to increase always had more proline than the stem and root. An with the increasing NaCl concentration upto 300 mM and at increasing accumulation of proline was found with increasing higher concentrations of NaCl, there was a decrease in the concentrations of NaCl. The accumulation of proline was chlorophyll content. A decrease in the chlorophyll content more in the leaf tissues than in the stem and root tissues of under salinity has been reported by several workers in a NaCl treated plants. The positive correlation exists between number of halophytes such as Avicennia sp. and Aegiceras the proline and salinity treatment. Salinity tolerance has been corniculatum (Shinde and Bhosale, 1985). The decrease of associated with the capacity of a species to accumulate proline chlorophyll is mainly attributed to the destruction of and it acts as an intracellular osmoticum. Proline is believed to chlorophyll a which is considered to be more sensitive to function as compatible solute in balancing cytoplasmic and salinity than chlorophyll b (Shinde and Bhosale, 1985). Similar results have been reported for other halophytes that vacuolar water potentials (Hassine et al., 2008). Generally salt International Journal of Research in Plant Science 2012; 2(1): 1-7

Fig.5.Effect of NaCl on chlorophyll a, chlorophyll b and total chlorophyll (mg g-1 fr. wt.) of Suaeda maritima on 60th day after treatment.

increased the chlorophyll content with increasing salinity upto an optimal level; Ipomoea pes-caprae (Venkatesan et al., 1995) and Ceriops roxburghiana (Rajesh et al., 1998). Effect of salinity on catalase Activity The effect of NaCl on the catalase activity in the leaves at various NaCl concentrations is presented in (Fig. 6). There was a steady increase in the enzyme activity with increasing salinity upto 300mM in Suaeda maritima at higher concentrations, the enzyme activity decreased. Enhanced activity of catalase was reported to be essential for the survival of the halophytes, Halimions portulacoides in natural saline habitats (Kalir and Poljak off-Mayber, 1981). The catalase activity increased with increasing concentration of NaCl upto optimum level in Ipomoea pes-caprae (Venkatesan and Chellappan, 1999). The catalase activity decreased with increasing concentration in Phaseolus radiatus (Saha and Gupta, 1999).

Effect of salinity on polyphenol oxidase activity The sodium chloride salinity enhanced the PPO activity upto the optimum level of 300 mM in Suaeda maritima and the data are given in (Fig. 6). Beyond, the optimum level, the PPO activity reduced gradually. Polyphenol oxidase activity increased with increasing salinity upto optimum concentration of 300 mM in Suaeda maritima. Increased polyphenol oxidase activity has been reported in halophytes such as Aegiceras corniculatum (Manikandan and Venkatesan, 2004). High polyphenol oxidase activity under stress indicates its ability to oxidize and to degrade the toxic substances such as phenolic compounds which are generally reported to be accumulated during salt stress (Subhashini and Reddy, 1990). Sharp increase in polyphenol oxidase activity under salinity stress was associated with enhanced rooting in Excoecaria agallocha, Cynometra iripa and Heritiera fomes (Basak et al., 2000). CONCLUSION In the present investigation, the effect of different concentration of sodium chloride on organic components, chlorophyll synthesis and activities of certain key enzymes of Suaeda maritima has been studied. Suaeda maritima could survive a wide range of 100-500 mM NaCl concentration. The upper limit for survival of this species to NaCl salinity was 500 mM. howere, favorable growth response by the seedlings was confined to 300 mM NaCl. REFERENCES

Fig.6. Effect of NaCl on catalase, peroxidase and polyphenol oxidase activity (units min-1 mg-1 protein.) in the leaf of Suaeda maritima on 60th day after treatment. Effect of salinity on peroxidase activity The peroxidase activity showed a similar increasing trend as that of catalyse upto the optimum level of NaCl salinity and data are given in (Fig. 6). The highest activity was recorded in Suaeda maritima at the optimum level (300mM NaCl). Even at the extreme salinity the peroxidase activity was equal to that of control. Increase in peroxidase activity indicated the formation of large amount of H2O2 and which could release enzyme from membrane structure (Zhang and Kirkham, 1994). Peroxidase is a scavenging enzyme which removes the toxic oxygen radicles from the cells. Significant increase in the peroxidase activity in the halophytes such as Aegiceras corniculatum (Manikandan and Venkatesan, 2004), in the salt tolerant varieties of spinach leaves (Oztiirk and Demir, 2003) and Xanthosoma sagittifolium (Kanmegne and omokoto, 2003). The increased peroxidase activity was mainly due to increased enzyme synthesis and might be useful for adaptation under conditions requiring prevention of peroxidation of membrane lipids (Kalir et al., 1984).

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