Lipid Peroxidation and Antioxidative Enzymes of Two Turfgrass Species Under Salinity Stress

Lipid Peroxidation and Antioxidative Enzymes of Two Turfgrass Species Under Salinity Stress

Pedosphere 23(2): 213–222, 2013 ISSN 1002-0160/CN 32-1315/P c 2013 Soil Science Society of China  Published by Elsevier B.V. and Science Press Lipid...

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Pedosphere 23(2): 213–222, 2013 ISSN 1002-0160/CN 32-1315/P c 2013 Soil Science Society of China  Published by Elsevier B.V. and Science Press

Lipid Peroxidation and Antioxidative Enzymes of Two Turfgrass Species Under Salinity Stress∗1 R. XU1 , M. YAMADA2 and H. FUJIYAMA2,∗2 1 United 2 Faculty

Graduate School of Agricultural Sciences, Tottori University, 4-101, Koyama-cho Minami, Tottori 680-8553 (Japan) of Agriculture, Tottori University, 4-101, Koyama-cho Minami, Tottori 680-8553 (Japan)

(Received June 18, 2012; revised January 21, 2013)

ABSTRACT Salinity stress is a major factor limiting the growth of turfgrass irrigated with recycled wastewater. The change in lipid peroxidation in terms of malondialdehyde (MDA) content and the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxide (APX) and glutathione reductase (GR) in the shoots and roots of Kentucky bluegrass and tall fescue were investigated under salinity stress. Plants were subjected to 0, 50, 100, 150 and 200 mmol L−1 NaCl for 40 d. The MDA content under salinity stress was lower in tall fescue than in Kentucky bluegrass in both shoots and roots. Activities of SOD in the shoots of both species increased with salinity stress. The activities of CAT and APX decreased in Kentucky bluegrass, but no significant difference in the activities of CAT and APX was observed in tall fescue. The activities of SOD, CAT and APX in the shoots of tall fescue were higher than those in Kentucky bluegrass. In the roots of Kentucky bluegrass, SOD and GR activities increased and CAT and APX activities decreased in comparison with the control. In the roots of tall fescue, salinity increased the activities of SOD, CAT, and APX. These results suggested that tall fescue exhibited a more effective protection mechanism and mitigated oxidative stress and lipid peroxidation by maintaining higher SOD, CAT and APX activities than Kentucky bluegrass. Key Words:

antioxidants, malondialdehyde, oxidative stress, recycled wastewater, salt tolerance

Citation: Xu, R., Yamada, M. and Fujiyama, H. 2013. Lipid peroxidation and antioxidative enzymes of two turfgrass species under salinity stress. Pedosphere. 23(2): 213–222.

INTRODUCTION Salinity is one of the most serious problems limiting plant growth and productivity. Approximately 6% of the earth’s land area (800 million hectares) is affected by either salinity or the associated condition of sodicity (FAO, 2006). High salt concentrations in soil cause ionic stress, hyperosmotic stress, and secondary stresses such as oxidative stress by increasing reactive oxygen species (ROS) including superoxide radicals (O− 2 ), hydrogen peroxides (H2 O2 ), and hydroxyl radicals (·OH) (Botella et al., 2005). Overproduction of ROS in mitochondria and chloroplasts under stress conditions has been suggested as the major contributor to oxidative damage in plant. In mitochondria, electron leakage and release of O− 2 and H2 O2 in respiration have been proposed. In chloroplast, many stress factors that limit CO2 assimilation due to stomatal closure lead to a decrease in carbon reduction by the Calvin cycle and to a decrease in oxidized NADPH+ , which can serve as an electron acceptor in photosyn∗1 Supported

thesis. Under these conditions that the availability of electron acceptors are limited to form photosystem I, oxygen can accept electrons to form O− 2 by the Mehler reaction, which triggers chain reactions that generate H2 O2 and ·OH (Perl-Treves and Perl, 2002). These ROS are highly reactive and can alter normal cellular metabolism through the oxidation of lipids, proteins and nucleic acids (Imlay, 2003). To mitigate the oxidative damage induced by ROS, plants employ a variety of enzymatic and non-enzymatic antioxidant defenses (Apel and Hirt, 2004). Enzymatic antioxidant systems typically consist of several antioxidant enzymes that participate in the detoxification of ROS. Superoxide dismutase (SOD) is a major scavenger of O− 2 , and its enzymatic action results in the formation of H2 O2 and O2 (Bowler et al., 1992). H2 O2 is scavenged by catalase (CAT) and ascorbate peroxidase (APX). CAT dismutates H2 O2 into H2 O and O2 , while APX, together with monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase (GR), converts H2 O2 to H2 O via the ascor-

by the Global Center of Excellence for Dryland Science from the Ministry of Education, Science, Culture, Sports and Technology of Japan. ∗2 Corresponding author. E-mail: [email protected].

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bate-glutathione pathway. The ascorbate-glutathione pathway provides protection against oxidative stress by a series of coupled redox reactions in photosynthetic tissues, mitochondria and peroxisomes (Foyer and Halliwell, 1976; Jim´enez et al., 1997). APX is the first enzyme in this pathway involved with the elimination of H2 O2 using ascorbate as an electron donor in an oxidation-reduction reaction (Noctor and Foyer, 1998). GR is the final enzyme in this pathway, which plays a crucial role in protecting plants against oxidative stress by maintaining reduced glutathione (GSH) (Asada, 1999; Blokhina et al., 2002). The elevated levels of GR activity could increase the GSH and oxidized glutathione (GSSG) ratio, which is required for ascorbate regeneration and activation of several CO2 fixing enzymes in the chloroplasts, ensuring NADP+ availability to accept electrons from the photosynthetic electron transport chain (de Azevedo Neto et al., 2006). The levels of malondialdehyde (MDA), which is the decomposition product of the polyunsaturated fatty acids in biomembranes, is widely used to measure the extent of lipid peroxidation and as an indicator of oxidative stress (Gossett et al., 1994; Hern´andez and Almansa, 2002). Thus, MDA tends to accumulate under salinity stress and has widely been used to distinguish between salt-tolerant and salt-sensitive cultivars (Luna et al., 2000; Hern´andez and Almansa, 2002; Meloni et al., 2003). Salinity stress of turfgrass is becoming prevalent because of the increase in using wastewater containing salts for turfgrass irrigation. The need for salttolerant turfgrass cultivars has substantially increased in recent decades (Marcum, 2006). Kentucky bluegrass (KBG; Poa pratensis L.), a salt-sensitive grass species with an average threshold electrical conductivity (EC) of 3 dS m−1 (Carrow and Duncan, 1998), is widely used for lawn, golf turf (except greens), athletic fields, and other general-purpose turfs in subarctic and temperate regions (Turgeon, 2008). Tall fescue (TF; Festuca arundinacea Schreb.), a moderately salt-tolerant species with an EC tolerance of 6–10 dS m−1 (Harivandi, 1988), is becoming an increasingly important lawn species and is widely used as a utility turfgrass in both warm and cool subtropical climates (Turgeon, 2008). Several studies have been conducted to assess the effect of salinity on KBG and TF (Horst and Beadle, 1984; Qian et al., 2001; Suplick-Ploense et al., 2002). In addition, the oxidative stress induced by abiotic factors, such as heat, low light, waterlogging and drought on turfgrass, and the role of antioxidant ac-

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tivity in stress tolerance have also been investigated (Zhang and Schmidt, 1999; Jiang et al., 2005; Xu et al., 2006; Wang and Jiang, 2007). A correlation between the resistance to salinity stress and efficient antioxidant defense systems was reported in numerous crop species, such as rice (Demirel and T¨ urkan, 2005), cotton (Gossett et al., 1994), beet (Bor et al., 2003), pea (Hern´ andez et al., 2000), and barley (P´erez-L´opez et al., 2009, 2010). However, studies on the effectiveness of antioxidant defense systems in the salinity tolerance of turfgrass have been limited. The objective of this study was to examine the changes in growth parameters, the level of lipid peroxidation, and the activities of antioxidant enzymes in the shoots and roots of two cool-season turfgrass species, KBG and TF, under salinity stress. Since no detailed investigations have been conducted on this topic to date, the information presented here will not only provide criteria for selecting salt-tolerant species and cultivars, but also for improving salt-tolerant turfgrass. MATERIALS AND METHODS Plant materials and salt stress treatments Seeds of KBG cv. Blue Star and TF cv. Little Hero were sterilized in 0.5% (w/v) sodium hypochlorite and placed on a magnetic stirrer for 10 min before being thoroughly rinsed with distilled water and sown in trays containing vermiculite. The air temperature and relative humidity in the growth chamber conditions were maintained at 24±2 ◦ C and 60%, respectively. Three-week-old seedlings, uniform in size and development, were transplanted to 4 L plastic pots (five plants per pot) filled with a nutrient solution in a glasshouse. The composition of the nutrient solution was as follows: 2.0 mmol L−1 N (NH4 NO3 ), 0.4 mmol L−1 P (NaH2 PO4 ), 2.0 mmol L−1 K (KCl), 1.0 mmol L−1 Ca (CaCl2 ·2H2 O), and 2.0 mmol L−1 Mg (MgSO4 ·7H2 O) for the macronutrients, and 2.0 mg L−1 Fe (FeSO4 ·7H2 O), 0.5 mg L−1 Mn (MnSO4 ·5H2 O), 0.2 mg L−1 B (H3 BO3 ), 0.1 mg L−1 Zn (ZnSO4 ·7H2 O), 0.01 mg L−1 Cu (CuSO4 ·7H2 O), and 0.05 mg L−1 Mo [(NH4 )6 Mo7 O24 ·4H2 O] for the micronutrients. The pH of the solution was adjusted to 6.5 by adding H2 SO4 or NaOH. The solution was aerated constantly and replaced twice a week throughout the experiment. The temperature and relative humidity in the glasshouse were 20–32 ◦ C and 60%–70% during the experiment, respectively.

ANTIOXIDATIVE RESPONSE OF TURFGRASS TO SALINITY

Plants were cultured under non-saline conditions for 15 d to ensure full establishment before starting salinity treatments. The nutrient solution was salinized with NaCl to 50, 100, 150 and 200 mmol L−1 . To avoid osmotic shock, salt concentrations were increased at 50 mmol L−1 increments over a 24 h period until the desired salinity level was reached. Nutrient solution without NaCl (0 mmol L−1 NaCl) was prepared for the control treatment. Salinity treatment lasted 40 d. Growth parameter measurements During the salinity treatment period, the plant growth parameters of shoot height, root length, dry weight (DW) of shoots and roots, turf quality, and leaf firing were determined. Shoots and roots were washed with deionized water and dried at 70 ◦ C for 48 h to determine DW. Turf quality and leaf firing were visually estimated weekly as described by Alshammary et al. (2004). Turf quality was estimated based on a scale of 1–9, with 9 being green, dense and uniform, 1 being thin and completely brown, and 6 being the minimum acceptable level. Leaf firing was estimated as the total percentage of chlorotic leaf area, with 0% indicating no leaf firing and 100% indicating totally brown leaves. Determination of lipid peroxidation Lipid peroxidation was determined in terms of MDA content using the thiobarbituric acid (TBA) method described by Heath and Packer (1968). Shoot and root samples were homogenized in 5 mL of 0.5% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 10 000 × g for 15 min. For every 1 mL aliquot of supernatant, 4 mL of 20% (w/v) TCA solution containing 0.5% (w/v) TBA was added. The mixture was heated to 95 ◦ C in a shaking waterbath for 30 min and then quickly cooled in an ice bath. After centrifuging at 12 000 × g for 15 min, the absorbance of the supernatant was read at 532 nm. The measurements were corrected for non-specific turbidity by subtracting the absorbance at 600 nm. The MDA content was calculated using an extinction coefficient of 155 mmol L−1 cm−1 . Extraction and assay of antioxidative enzymes Fresh shoot and root samples were collected from both species after salinity treatment for enzyme analysis. The samples were frozen in liquid nitrogen immediately after harvesting and stored at −70 ◦ C until enzyme assays were performed. For protein and enzymes extractions, samples (0.15 g of shoot and 0.2 g of root) were ground to a powder using a mor-

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tar and pestle pre-cooled with liquid nitrogen and homogenized in 50 mmol L−1 potassium phosphate buffer (pH 7.8) containing 1 mmol L−1 ethylenediaminetetraacetic acid (EDTA), 1 mmol L−1 ascorbate, and 2% (w/v) polyvinylpolypyrrolidone. Homogenates were then centrifuged at 20 000 × g for 30 min at 4 ◦ C and supernatants were divided into two parts. One part was used directly for CAT, APX and GR assays, while the other was transferred to a dialysis membrane (Wako, Osaka, Japan) and dialyzed at 4 ◦ C in 10 mmol L−1 potassium phosphate buffer (pH 7.8) for 12 h (buffer was changed at 3 h intervals). After centrifuging the dialyzed solution at 13 000 × g for 20 min, the supernatant was used for the SOD assay and total protein determination. SOD was assayed according to Tanaka and Sugahara (1980). The reaction was performed in a total volume of 1 mL containing 50 mmol L−1 potassium phosphate buffer (pH 7.8, containing 0.1 mmol L−1 EDTA), 0.1 mmol L−1 cytochrome c, 0.1 mmol L−1 xanthine, enzyme extract, and 0.3 unit mL−1 of xanthine oxidase. The reaction was initiated by the addition of xanthine oxidase and the absorbance was measured at 550 nm. One unit of SOD was defined as the amount of enzyme that inhibits the rate of cytochrome c reduction by 50%. CAT activity was measured using the method of Aebi (1984) with slight modifications. Disappearance of H2 O2 was monitored by measuring the decrease in absorbance at 240 nm. The reaction was carried out in a solution containing 1 mmol L−1 EDTA in 50 mmol L−1 potassium phosphate buffer (pH 7.8), 5 mmol L−1 H2 O2 , and enzyme extract in a total volume of 1 mL. CAT activity was calculated using an extinction coefficient of 0.04 mmol L−1 cm−1 and one unit of CAT activity was defined as the amount of enzyme required to decompose 1 μmol of H2 O2 per minute. APX activity was determined by measuring the decrease in absorbance at 290 nm (Nakano and Asada, 1981). The assay was performed in a 1 mL reaction mixture containing 1 mmol L−1 EDTA in 50 mmol L−1 potassium phosphate buffer (pH 7.8), 0.5 mmol L−1 ascorbate, the enzyme extract, and 0.5 mmol L−1 H2 O2 . The concentration of oxidized ascorbate was calculated using an extinction coefficient of 2.8 mmol L−1 cm−1 and one unit of APX activity was defined as the amount of enzyme necessary to catalyze the oxidation of 1 μmol ascorbate per minute. GR activity was assayed by measuring the decrease in absorbance at 340 nm (Tanaka et al., 1988). The assay mixture contained 1 mmol L−1 EDTA in 50 mmol L−1 potassium phosphate buffer (pH 7.8), 0.1

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mmol L−1 NADPH, enzyme extract, and 0.1 mmol L−1 GSSG in a total volume of 1 mL. The reaction was initiated by adding GSSG and the NADPH oxidation rate was monitored at 340 nm. GR activity was calculated using an extinction coefficient of 6.2 mmol L−1 cm−1 for NADPH and one unit of enzyme was defined as the amount of enzyme required to oxidize 1 μmol of NADPH per minute. The activities of the enzymes were expressed as units mg−1 protein. Total soluble protein content was determined using bovine serum albumin as a standard (Bradford, 1976). Statistical analyses The experiment was set up as a completely randomized design including two turfgrass species and five salinity levels. The effects of turfgrass species, salinity levels and their interactions on measured variables were assessed by a two-way analysis of variance (ANOVA) using SPSS version 10.0J software for Windows (SPSS, SPSS Japan Inc., Japan). Mean values were compared by Duncan’s multiple range test when significant differences occurred at 5% level. RESULTS Growth parameters The shoot height, root length, shoot DW and root DW of KBG decreased significantly with increasing

NaCl concentrations (Table I). Although the shoot height and shoot DW of TF decreased with increasing salinity, TF exhibited the higher shoot height and shoot DW than KBG at the same salinity stress. The root length and root DW of TF were not significantly different in 100, 150 and 200 mmol L−1 NaCl treatments. Although turf quality declined as salinity increased in both species (Table I), the turf quality of TF was higher during the experimental period. Turf quality of KBG decreased to 3.88 at 100 mmol L−1 NaCl, but TF maintained a minimal acceptable quality (6.69) at 100 mmol L−1 NaCl. Leaf firing in both species increased with an increase in salinity, reaching 52% in KBG and 28% in TF at 100 mmol L−1 NaCl. Leaf firing in KBG was markedly higher than that in TF at the same salinity levels. The results of growth parameters (Table I) indicated that growth inhibition by NaCl was more severe in KBG than in TF. Lipid peroxidation Lipid peroxidation was determined in terms of MDA content. The NaCl concentration and turfgrass species were highly significant factors for the MDA content in both shoots and roots (Table II). The MDA content increased with increasing salinity in both turfgrass species (Fig. 1). The accumulation of MDA in the shoots and roots of KBG was higher than that observed in TF, indicating that the increase in lipid peroxidation induced by NaCl stress was higher in KBG than in TF.

TABLE I Effect of salinity on plant growth parameters in two turfgrass species, Kentucky bluegrass (KBG) and tall fescue (TF) Turfgrass species KBG

TF

NaCl Species (S) S × NaCl

NaCl mmol 0 50 100 150 200 0 50 100 150 200

Shoot height L−1

Root length

cm 32.20±1.05a) ab) 27.42±0.49b 22.40±0.41c 20.70±0.46c 18.03±0.66d 40.84±0.69a 36.26±0.29b 30.39±0.83c 25.75±1.09d 22.88±0.81e

31.59±0.65a 24.90±0.64b 22.61±0.61c 19.57±0.33d 14.86±0.52e 34.37±0.78a 33.90±1.51a 39.48±2.63b 29.99±0.93a 29.47±1.14a

162.0*** 236.5*** 9.9*

29.6*** 212.1*** 10.9***

Shoot dry weight g 2.49±0.13a 1.65±0.09b 1.25±0.09c 0.96±0.04d 0.57±0.03e 2.83±0.10a 2.20±0.36b 1.32±0.07c 1.36±0.10c 1.14±0.05c F value 54.5*** 19.6*** 1.0NSc)

Root dry weight

Turf quality

Leaf firing

0.76±0.04a 0.65±0.03b 0.46±0.02c 0.29±0.004d 0.15±0.01e 0.69±0.01a 0.67±0.04a 0.59±0.01b 0.54±0.01b 0.51±0.04b

8.62±0.08a 6.25±0.16b 3.88±0.08c 2.70±0.12d 2.27±0.11e 8.80±0.10a 8.13±0.08b 6.69±0.26c 5.68±0.15d 4.32±0.10e

% 13.52±0.11e 32.87±1.19d 56.71±1.65c 74.32±1.95b 84.24±1.74a 9.33±0.15e 15.51±0.69d 29.54±1.38c 56.13±1.16b 70.28±0.55a

88.9*** 80.5*** 24.5***

542.0*** 544.6*** 34.2***

999.6*** 434.2*** 22.8***

plant−1

*, ***Significant at P = 0.05 and P = 0.001 levels, respectively. a) Means±standard errors (n = 5). b) Means followed by the same letter(s) within a column for each turfgrass species are not significantly different at P < 0.05 by Duncan’s multiple range test. c) Not significant.

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TABLE II

Activities of antioxidative enzymes

F values of ananlysis of variance of NaCl, species, and their interaction for malondialdehyde (MDA) content and antioxidant activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxide (APX) and glutathione reductase (GR)

The SOD activities in the shoots of both turfgrass species and the activities in the roots of TF increased under NaCl stress, whereas SOD activities in the roots of KBG were not significantly different with increasing salinity (Fig. 2). The SOD activities in the shoots and roots of TF were higher than those in KBG, especially at higher salinities. The results of ANOVA exhibited that NaCl and turfgrass species had significant effects on SOD activities (Table II). However, the increase of SOD activities in TF under NaCl treatment was larger than that observed in KBG. Compared to the control, the SOD activities at 200 mmol L−1 NaCl increased by 114.6% for shoots and 87.6% for roots of TF, while SOD activities in the shoots and roots of KBG increased by 21.4% and 11%, respectively. CAT activities in the shoots and roots of KBG were

Tissue

Item

NaCl

Species (S)

NaCl × S

Shoot

MDA SOD CAT APX GR MDA SOD CAT APX GR

84.75*** 19.86*** 9.00*** 0.31NSa) 51.33*** 264.67*** 25.02*** 2.80* 12.06*** 4.83**

137.81*** 18.50*** 111.23*** 240.92*** 79.10*** 345.44*** 120.25*** 47.49*** 16.36** 1.82NS

11.22*** 6.19** 1.30NS 2.32NS 15.52*** 40.79*** 14.24*** 5.54** 17.52*** 17.76***

Root

*, **, ***Significant at P = 0.05, P = 0.01, and P = 0.001 levels, respectively. a) Not significant.

Fig. 1 Malondialdehyde (MDA) contents in the shoots and roots of Kentucky bluegrass (KBG) and tall fescue (TF) under different NaCl treatments. Vertical bars indicate standard errors of the means (n = 3). Bars with the same letter(s) for each turfgrass species are not significantly different at P < 0.05 by Duncan’s multiple range test.

Fig. 2 Superoxide dismutase (SOD) activities in the shoots and roots of Kentucky bluegrass (KBG) and tall fescue (TF) under different NaCl treatments. Vertical bars indicate standard errors of the means (n = 3). Bars with the same letter(s) for each turfgrass species are not significantly different at P < 0.05 by Duncan’s multiple range test.

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reduced with an increase in NaCl concentrations (Fig. 3). The CAT activities in the shoots of TF were higher in the control than in salinity conditions; however, CAT activities did not change between 50 and 200 mmol L−1 NaCl treatments (Fig. 3). The CAT activities in the roots of TF were higher in salt-stressed plants than in the control plants. The CAT activities in the shoots and roots of KBG were significantly lower than those in TF for all salinity treatments. The results of ANOVA indicated that NaCl and turfgrass species had significant effects on CAT activities (Table II). APX activities did not change significantly in the shoots of KBG, and decreased with increasing salinity in the roots of KBG (Fig. 4). At 200 mmol L−1 NaCl, root CAT activities in KBG decreased to 56% of the control. Activities of APX in the shoots of TF did not show a significant change under salt stress. However,

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the activities of APX in the roots of TF were higher under salt treatments compared to the control. The APX activities of both shoots and roots were significantly lower for KBG than for TF under salt stress (Fig. 4). NaCl affected APX activities in the roots, but not in the shoots, according to the results of ANOVA (Table II). Compared with the control, the activities of GR in the shoots of KBG and TF were decreased with NaCl stress (Fig. 5). However, there was no significant difference in the GR activities of plants subjected to 100 to 200 mmol L−1 NaCl in both turfgrass species. The ANOVA showed that NaCl and turfgrass species had significant effects on GR activities in the shoots (Table II). GR activity in TF roots was highest at 50 mmol L−1 NaCl and there was no significant difference in the 100 to 200 mmol L−1 NaCl treatments. Root GR activities in KBG increased gradually with an increase

Fig. 3 Catalase (CAT) activities in the shoots and roots of Kentucky bluegrass (KBG) and tall fescue (TF) under different NaCl treatments. Vertical bars indicate standard errors of the means (n = 3). Bars with the same letter(s) for each turfgrass species are not significantly different at P < 0.05 by Duncan’s multiple range test.

Fig. 4 Ascorbate peroxidase (APX) activities in the shoots and roots of Kentucky bluegrass (KBG) and tall fescue (TF) under different NaCl treatments. Vertical bars indicate standard errors of the means (n = 3). Bars with the same letter(s) for each turfgrass species are not significantly different at P < 0.05 by Duncan’s multiple range test.

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Fig. 5 Glutathione reductase (GR) activities in the shoots and roots of Kentucky bluegrass (KBG) and tall fescue (TF) under different NaCl treatments. Vertical bars indicate standard errors of the means (n = 3). Bars with the same letter(s) for each turfgrass species are not significantly different at P < 0.05 by Duncan’s multiple range test.

in salinity and were higher than those in TF at 100, 150 and 200 mmol L−1 NaCl (Fig. 5); however, turfgrass species were not significant factors on GR activities in the roots (Table II). DISCUSSION Plant species differ greatly in their growth response to salinity. It is important to study the physiological characterization of plants to salinity stress to define relative salinity tolerance. Parameters, such as shoot growth, root mass, root length, and turf quality are well suited for examining salinity tolerance in turfgrass species (Alshammary et al., 2004). In this study, the growth parameters of root length, root DW, leaf firing and turf quality revealed that TF was more salt tolerant than KBG, corroborating the findings of Alshammary et al. (2004) and Marcum (2008). Shoot growth in both species decreased significantly with increasing salinity (Table I). Marcum (2008) reported that shoot growth of salt-sensitive to moderately-tolerant turfgrass species declined linearly with increased salinity stress. However, TF exhibited high root length and root DW, which resulted in increased root/shoot ratios under salt stress. The increase in root/shoot ratio of TF can maintain an optimal water balance between the root water absorption and shoot transpirational area. An inability to adjust the root/shoot ratio in KBG may explain the relatively poor salinity tolerance in this species. The suppression of turfgrass growth due to salinity stress was manifested as a decrease in turf quality and an increase in leaf firing. Both turf quality and leaf firing were significantly affected by increasing salinity. Compared to their respective controls, the decrease in

turf quality and increase in leaf firing under salinity stress were larger for KBG than for TF, indicating that TF was more tolerant to salinity than KBG. Salinity led to stunted growth through reducing leaf area and photosynthetic rates, primarily due to stomatal limitation. Lowering of intercellular CO2 fixation rate can cause oxidative damage due to increased ROS generation. To keep ROS under control, plants have evolved an efficient antioxidant defense system. In the present study, the changes of MDA content and enzyme activities of SOD, CAT, APX and GR suggest that oxidative stress is an important component of salt stress in KBG and TF. The MDA content increased with increasing salinity in the shoots and roots of both turfgrass species (Fig. 1), indicating that cell membranes were damaged in both turfgrass species. Growth inhibition, decreased turf quality, and increased leaf firing are associated with cell membrane damage due to salinity-induced lipid peroxidation (Huang et al., 2001). The MDA content in TF was significantly lower than that in KBG under salt stress, indicating that less lipid peroxidation occurred in response to the higher activities of SOD, CAT, and APX observed in TF. In KBG, a significant increase in MDA levels appeared to be correlated with a decrease in the activities of CAT and APX under salt stress, with SOD activity in the root remaining unchanged. The higher MDA content in KBG implied that the oxidative damage was more severe in KBG and that the antioxidant defense mechanisms of KBG were less effective than those of TF; similar evidence of lipid peroxidation and antioxidant activity has been reported by other researchers (Shalata and Tal, 1998; Ben Amor et al., 2006; P´erez-L´opez et al., 2009; Seckin et al., 2010).

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In the antioxidative systems of plants, SOD plays a central role in the enzymatic defense system by catalyzing the dismutation of O− 2 to H2 O2 and O2 (Bowler et al., 1992). In our study, SOD activities increased significantly under conditions of salinity stress in the shoots of both species and in the roots of TF. The increases of SOD activities in the shoots and roots of TF were larger than those of KBG, implying that TF has better O− 2 radical scavenging ability. In addition to being an important agent of cellular toxicity, the product of SOD activity, H2 O2 , is also an important signal molecule between environmental stress and adaptive response (Foyer and Noctor, 2003; Seckin et al., 2010). In plants, CAT and APX are considered to be the most important enzymes regulating intercellular levels of H2 O2 . CAT is primarily localized in peroxisomes and glyoxysomes where it breaks down H2 O2 into water and O2 . Ben Amor et al. (2007) reported that H2 O2 accumulation under salinity stress was related to a decrease in CAT activity. Our data showed that CAT activity decreased in both shoots and roots of KBG under salinity stress. However, CAT activity remained unchanged in the shoots and roots of TF, indicating that the scavenging of H2 O2 by TF is more effective than that by KBG. Like CAT, APX plays a key role in protecting the plant against oxidative stress by scavenging H2 O2 in the chloroplasts, cytosol, mitochondria, and peroxisome of cells (Asada, 2006). In the present study, TF exhibited higher APX activities than KBG under salinity stress. We also found that the increase in APX activity was accompanied by higher CAT activities in TF, implying that TF has a more effective H2 O2 scavenging mechanism than KBG. The activity of GR, which catalyzes the NADPHdependent reduction of GSSG, is important in combating oxidative stress arising from abiotic stress (Foyer et al., 1991). Several researchers have suggested that a salt-induced increase in GR activity is more prevalent in salt-tolerant cultivars than in salt-sensitive cultivars (Hern´ andez et al., 2000; Meloni et al., 2003; Demiral and T¨ urkan, 2005). However, GR activity decreased in the shoots of both turfgrass species under salt stress in this study. Our findings are in agreement with those of Shalata and Tal (1998), who reported a decrease in GR activity in the leaves of both salt-tolerant and saltsensitive tomato plants under salt stress. Interestingly, GR activity increased with the increasing NaCl concentrations in the roots of KBG (Fig. 5), being greater in KBG than in TF at NaCl concentrations of 100, 150 and 200 mmol L−1 . The reduced level of GR activities in shoots of KBG and TF could decrease the

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GSH/GSSG ratios. Gechev et al. (2005) reported that cell growth arrest and blocked cell division are associated with low GSH/GSSG ratio and GSH depletion. The transgenic tobacco plants overexpressing GR had both high levels of GSH and increased tolerance to oxidative stress (Broadbent et al., 1995). Therefore, GR may be a key enzyme for development of salt-tolerant turfgrass cultivars. In the present study, the results of plant growth, turf quality and leaf firing indicated that TF was more tolerant to salinity stress than KBG. Regarding the salt-induced increase in MDA content, our research demonstrated that membrane lipid peroxidation occurred in both turfgrass species. Compared to KBG, the lower level of lipid peroxidation observed in TF suggested that TF had the more effective antioxidant defense system. The increased SOD activity in TF, which corresponded to increased CAT and APX activities, played a central role in scavenging O− 2 and H2 O2 . The high SOD activity in the shoots of KBG indicated that H2 O2 was produced from the conversion of O− 2. However, compared to the control plants, the relatively lower activities of CAT, APX and GR in salt-stressed KBG shoots indicated that H2 O2 scavenging was less effective in the salt-stressed plants. In fact, this surplus H2 O2 may be the main reason for the extensive lipid peroxidation and growth inhibition observed in KBG. To the best of our knowledge, this is the first report to demonstrate the changes in the antioxidant systems of the cool-season turfgrasses, KBG and TF, under salinity stress. Our results demonstrated that salinity stress had a significant impact on the antioxidative enzymes in both turfgrass species. KBG, a salt sensitive cool-season turfgrass, lost ROS scavenging ability under salt stress. Although TF, a moderately salinity-tolerant turfgrass species, could eliminate ROS by increasing antioxidative enzyme activity, the observed decrease in the DW and turf quality of TF, as well as increased leaf firing and MDA content under salinity stress, indicated that the antioxidant defense system in TF could not prevent cell injury completely. CONCLUSIONS Salinity stress had effect on plant growth parameters, MDA content and antioxidant activities in KBG and TF. The differences in antioxidative enzyme activities of shoots and roots may explain the salt tolerance of KBG and TF. TF exhibited a better antioxidant defense system against oxidative stress and lipid peroxidation by maintaining higher SOD, CAT and APX

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