Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIII, 2012

DETERMINATION OF ANTIOXIDANT ENZYMATIC ACTIVITY IN SEVERAL HALOPHYTES FROM DOBROGEA AREA

MIHAELA AURELIA IVAN1, MARIA-MAGDALENA ZAMFIRACHE2, MARIUS-NICUOR GRIGORE3, LCRMIOARA OPRIC4*
Keywords: halophytes, Plantago sp., Limonium gmelini, Bassia sedoides, Spergularia media, superoxide dismutase, catalase, peroxidase activities. Abstract. Halophytes have evolved various mechanisms of adaptations to stress tolerance including an increase of antioxidant enzymes activities. The present study was conducted in order to investigate the activity of some antioxidant enzymes (superoxide dismutase SOD, catalase CAT and peroxidase POD) in several halophytes. For this, four halophytic species were collected during summer of 2012 from two distinct saline areas, located in South-East of Romania (Dobrogea). Species collected from Histria are Plantago maritima and Bassia sedoides; the first mentioned species was collected in various stages of development (vegetative and flowering phases). Species Plantago coronopus, Spergularia media, Limonium gmelini, and Bassia sedoides from Sulina were collected from two different habitats: littoral area and an habitat located at 1000 m from littoral. The results show that halophytes collected from 1000 m to littoral area were characterized by higher levels of SOD activity than those collected from littoral. The peroxidase activity in halophytes collected from Sulina show various responses according to species and collecting points. Some of halophytes collected from Histria and Sulina have an undetectable level of catalase activity at the moment of determination; perhaps the role of this enzyme for removing H2O2 has been taken by peroxidase.

Littoral environment is a naturally developed ecosystem which is severely vulnerable and susceptible to water pollution and climate changes (Mahlagha et al., 2010). Halophilic (salt-loving) plants, or halophytes, are salt-tolerant plants specific for saline environments (Grigore et al., 2012). Known as halophytes, littoral plants naturally grow in salty littoral ecosystems. Generally, halophytes include large taxonomic varieties that occupy diverse habitats, from extremely dry to temporarily waterlogged sites or salt marshes. Some of these species which are organized in different levels continue their life and growth cycle under such a difficult conditions (Tipirdamaz et al., 2005; Flowers et al., 1986). Halophyte species are recognized as high biopotential plants (Glenn et al.,1999; OLeary, 1986). These plants are capable of growing and reproducing in saline conditions, as groups have several physiological adaptations that facilitate their survival in saline environments. These species are highly evolved and specialized organisms with well adapted morphological, anatomical, and physiological characteristics allowing them to proliferate in soils possessing high salt concentrations (Flowers et al.,1977; Flowers and Colmer, 2008). The plants ability to adapt at saline environmental medium could be a consequance of many different physiological adjusments that operate through various pathwys. Plant responses to salt stress are complex, extremely variable, mutually linked, and include a wide range of effects at the molecular, cellular, tissue and whole-plant level (Madhava Rao et al.,2006). Thus, the halophytic plants present many changes upon exposure to environmental stresses leading to metabolic disturbance and finally generation of reactive oxygen species (ROS). Oxidative stress is an additional phenomenon of stress impact on plants. Thus, salt stress produces secondary oxidant stress on plants. This secondary effect emerges as a consequence of hyperosmolarity caused by imposing of plants to salt stress or drought conditions, resulting in appearance of the reactive oxygen molecules, such as hydrogen peroxide, hydroxyl radicals and superoxide anions (Xiong et al.,2002). In plants, ROS are continuously produced predominantly in chloroplasts, mitochondria, and peroxisomes. In the absence of protective mechanism these very toxic ROS cause in plants oxidative damage to protein, DNA and lipids (Mittler et al.,2010). ROS act as signals for the activation of stress-response and defense pathways. Major ROS scavenging mechanism of plants include antioxidative enzymes (superoxide dismutase, catalase and peroxidase) which are key elements of the defense mechanisms. Most of the halophytes have shown increases efficiency of antioxidant enzyme machinery thus removing the ROS levels to a greater extent and maintain the plants survival under stressful conditions. For this, halophytes have evolved various mechanisms of adaptations to stress tolerance including an increase of antioxidant enzymes activities. Correlations between antioxidant capacity and salt tolerance have been suggested in a large number of true halophytes, such as Limonium bicolor (Li, 2008), Thellungiella halophila (Radyukina et al.,2007) and Suaeda salsa (Wang et al.,2008). The regulation of antioxidant enzyme activities may represent an important cellular mechanism for salt resistance.

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INTRODUCTION
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Mihaela Aurelia Ivan et al Determination of antioxidant enzymatic activity in several halophytes from Dobrogea area

The aim of this research was to study the behavioral pattern of several halophytes collected from Dobrogea area. Superoxide dismutase, catalase and peroxidase enzymes activities of these littoral plants were studied and some of their behavioural pattern was compared.

MATERIALS AND METHODS

Halophytic species were collected during summer of 2012 from two distinct saline areas, located in South-East of Romania (Dobrogea). Species collected from Histria are Plantago maritima and Bassia sedoides; the first mentioned species was collected in various stages of development (vegetative and flowering phases). Species Plantago coronopus, Spergularia media, Limonium gmelini, and Bassia sedoides from Sulina were collected from two different habitats: littoral area (Z I) and an habitat located at 1000 m from littoral (Z II). Extraction and estimation of enzyme activities For determination of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities it was used homogenized plantlets (0,3g) which disodium phosphate buffer (pH-7). Each homogenate was transferred to centrifuge tubes and was centrifuged at 40C for 20 min. at 3000xg. The supernatant was used for enzymes activity assay. The principle of SOD activity assay was based on the inhibition of nitroblue tetrazolium (NBT) reduction. Illumination of riboflavin in the presence of O2 and electron donor generates superoxide anions and this has been used as the basis of assay of SOD. The reduction of NBT by superoxide radicals to blue colored was followed at 560 nm. One unit of SOD activity is defined as that amount of enzyme required to inhibit the reduction of NBT by 50% under the specified conditions (Artenie et al, 2008). CAT activity was determined according to Sinha method based on reduction of dichromate in acetic acid to chromic acetate when heated in the presence of hydrogen peroxide than the chromic acetate was measured at 570 nm. One unit of CAT activity is defined as the enzyme amount that degrades one mmol H2O2/min. (Sinha, 1972). Determination of POD activity is based on the measurement of color intensity of o-dianisidine oxidation product with hydrogen peroxide in presence of peroxidase. Protein content was determined according to Bradford method and all enzymes concentration was reported as unit/mg protein.

Plants have evolved a complex regulatory network to mediate biotic and abiotic stress responses based on ROS synthesis, scavenging, and signaling (Pallavi Sharma et al., 2012). Halophytes show immense diversity in habitat and behavior to tolerate the abiotic stress conditions with uneven distribution across the taxa of flowering plants (Flowers et al., 2010). Toxic reactive oxygen species such as superoxide radicals, H2O2 and singlet oxygen are formed regularly in many cellular reactions when electrons are misdirected and donated to oxygen. Enzymes such as superoxide dismutases (SOD) react with superoxide radicals at almost diffusion limited rates to produce H2O2 which may be disposed by catalase and peroxidase. SOD is the rst line of defence against oxidative stress in plants, playing an important part in determining the concentration of anion superoxid and hydrogen peroxide. The balance between SOD and the different H2O2-scavenging enzymes in cells is considered to be crucial in determining the steady state level of superoxide radicals and hydrogen peroxide (Asada and Takahashi, 1987, Bowler et al., 1991). Generally, in response to salt stress condition, plants accumulate compatible solutes to maintain ion homeostasis (Ashraf and Foolad, 2007). Halophytes in particular, seem to be largely dependent on their capacity to compartmentalize toxic ions in the vacuole and to accumulate compatible solutes in the cytoplasm (Glenn et al.,1999). These compatible osmolytes act as osmoprotectant, directly by stabilizing proteins and membrane structures under dehydration conditions, and by protecting the cell against oxidative stress as scavengers of reactive oxygen species (ROS) (Szabados and Savour, 2010). Our results show that halophytes collected from 1000m to Sulina littoral area (Z II) were characterized by higher level of SOD activity (with exception of Spergularia media) than those collected from littoral (Z I) (Figure 1). According to some authors (Esfandiari et al, 2007, Israr and Sahi, 2006) the low SOD activity is concomitant with little damage to vital biomolecules.

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RESULTS AND DISCUSSIONS
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Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIII, 2012

The different stages of development (vegetative and flowering phases) of Plantago maritima collected from Histria area did not modify significantly the SOD activity. It appears that the activity of SOD under salinity varies depending upon plant species, its age, organ analyzed as well as the level of salinity (Chaparzadeh et al., 2004). Increased activity of SOD is often correlated with increased tolerance of the plant against environmental stresses (Sharma Pallavi et al., 2012).

H2O2 produced by SOD influence is detoxified by the two groups of enzymes catalases (CAT) and peroxidases (POD). CAT is suggested to be involved in mass scavenging of H2O2 whereas POD is suggested to be implicated in fine regulation of H2O2 (Mittler, 2002, Willekens H. et al., 1997). Among the antioxidant mechanisms developed by cells to maintain redox homeostasis, the CAT antioxidant enzyme is one of the most efficient in degrading hydrogen peroxide in water and oxygen (Lesser, 2006). Some of halophytes like Plantago maritima collected from Histria area or Plantago coronopus and Limonium gmelini from Sulina area have an undetectable level of CAT activity (Figure 2). That can be explained perhaps, by the presence of small amounts of hydrogen peroxide and thus the role of this enzyme being taking by peroxidase. H2O2 at low concentrations is suggested to play a role in signaling under stress (Apel and Hirt, 2004). Therefore, the role of H2O2-detoxifying enzymes is to impose tight control on its cellular concentrations rather than to remove it completely. In halophytes collected from Sulina area the peroxidase activity has indicate varied responses according to species and collecting points (Figure 3). Thus, generally the POD activity level of species from Sulina area was significant higher then those collected from Histria area. The increase of POD activity in these halophytes might be a consequence of the H2O2 formation

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Figure 1. Variation of superoxide dismutase activity in several halophytes collected from Dobrogea area (Z II = 1000m in Sulina littoral area, Z I = Sulina littoral area)

Mihaela Aurelia Ivan et al Determination of antioxidant enzymatic activity in several halophytes from Dobrogea area

in large amount due the different salinity level of collecting point. Higher POD activity was found in Plantago coronopus (Z II) and Spergularia media (Z I) collected from Sulina area while Plantago maritima and Basia sedoides from Histria area have lower level activity. High POD activity under stress indicates its ability to degrade the toxic substances such as H2O2 compounds which are generally reported to be accumulated during salt stress. The development stage of halophytes influenced the POD activity and this enzyme has activity reduced to half in Plantago maritima flowering phase than vegetative phase.

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Figure 2. Variation of catalase activity in several halophytes collected from Dobrogea area (Z II = 1000m in Sulina littoral area, Z I = Sulina littoral area)

Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIII, 2012

Figure 3. Variation of peroxidase activity in several halophytes collected from Dobrogea area (Z II = 1000m in Sulina littoral area, Z I = Sulina littoral area) CONCLUSION

Induction of antioxidant enzymes is an essential component of adaptive defense mechanism against salt stress in halophytes and protects them against ROS. Our results suggest that the changes of antioxidant enzymes activity of various halophytes from Histria and Sulina areas are related to the level of resistance to salinity being influenced by the collected area and the two different habitats as well as the different stages of development (vegetative and flowering phases). Thus the results show that halophytes collected from 1000 m to littoral area were characterized by higher levels of SOD activity than those collected from littoral. The peroxidase activity in halophytes collected from Sulina show various responses according to species and collecting points. Some of halophytes collected from Histria and Sulina have an undetectable level of catalase activity at the moment of determination; perhaps the role of this enzyme for removing H2O2 has been taken by peroxidase. REFERENCES

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