Enhancement of Superoxide Dismutase and Catalase Activities and Salt Tolerance of Euhalophyte Suaeda salsa L. by Mycorrhizal Fungus Glomus mosseae

Pedosphere 22(2): 217–224, 2012 ISSN 1002-0160/CN 32-1315/P c 2012 Soil Science Society of China  Published by Elsevier B.V. and Science Press

Enhancement of Superoxide Dismutase and Catalase Activities and Salt Tolerance of Euhalophyte Suaeda salsa L. by Mycorrhizal Fungus Glomus mosseae∗1 LI Tao1 , LIU Run-Jin2 , HE Xin-Hua3,4 and WANG Bao-Shan1,∗2 1

Key Laboratory of Plant Stress Research, College of Life Sciences, Shandong Normal University, Jinan 250014 (China) Institute of Mycorrhizal Biotechnology, Qingdao Agricultural University, Qingdao 266109 (China) 3 School of Plant Biology, University of Western Australia, Crawley, WA 6009 (Australia) 4 Northern Research Station, USDA Forest Service and School of Forest Resources and Environmental Science, Michigan Technological University, Houghton, MI 49931 (USA) 2

(Received April 30, 2011; revised November 14, 2011)

ABSTRACT Arbuscular mycorrhizal (AM)-mediated plant physiological activities could contribute to plant salt tolerance. However, the biochemical mechanism by which AM fungi enhance salt tolerance of halophytic plants is unclear. A pot experiment was conducted to determine whether salt tolerance of the C3 halophyte Suaeda salsa was enhanced by the AM fungus Glomus mosseae. When 60-day-old S. salsa seedlings were subjected to 400 mmol L−1 NaCl stress for 35 days, plant height, number of leaves and branches, shoot and root biomass, and root length of G. mosseae-colonized seedlings were significantly greater than those of the nonmycorrizal seedlings. Leaf superoxide dismutase (SOD) activity at all sampling times (weekly for 35 days after salt stress was initiated) and leaf catalase (CAT) activity at 2 and 3 weeks after salt stress was initiated were also significantly enhanced in G. mosseae-colonized S. salsa seedlings, while the content of leaf malondialdehyde (MDA), a product of membrane lipid peroxidation, was significantly reduced, indicating an alleviation of oxidative damage. The corresponding leaf isoenzymes of SOD (Fe-SOD, Cu/Zn-SOD1, and Cu/Zn-SOD2) and CAT (CAT1 and CAT2) were also significantly increased in the mycorrhizal seedlings after 14 days of 400 mmol L−1 NaCl stress. Our results suggested that G. mosseae increased salt tolerance by increasing SOD and CAT activities and forming SOD and CAT isoforms in S. salsa seedlings. Key Words:

antioxidant enzymes, isoenzyme, malondialdehyde, NaCl tolerance, oxidative stress

Citation: Li, T., Liu, R. J., He, X. H. and Wang, B. S. 2012. Enhancement of superoxide dismutase and catalase activities and salt tolerance of euhalophyte Suaeda salsa L. by mycorrhizal fungus Glomus mosseae. Pedosphere. 22(2): 217–224.

INTRODUCTION More than 6% (> 800 million ha) of the total land area and 20% (230 million ha) of irrigated lands in the world are salt-affected soils (Munns and Tester, 2008). In general, saline soils (≥ 40 mmol L−1 NaCl) reduce the growth of non-halophytes, which include almost all major grain crops, and prevent non-halophytes from completing their life cycles (Flowers and Colmer, 2008; Munns and Tester, 2008). Therefore, saline soils are a main threat to global crop production. The di∗1

rect impact of salt on plant growth could be due to the decrease in osmotic potential in the soil solution, the toxicity caused by excessive Na+ and Cl− in plant cells, and/or the nutrient imbalance in plant tissues (Evelin et al., 2009). Improving saline soils and alleviating plant salt stress have been major challenges in sustainable agriculture around the world. On the other hand, halophytes, which constitute about 1% of the world flora, grow naturally in saline soils and are able to tolerate and complete their life cycles under high levels (100 to

Supported by the National High Technology Research and Development Program (863 Program) of China (No. 2007AA091701) and the National Natural Science Foundation of China (No. 30870138). ∗2 Corresponding author. E-mail: [email protected].

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200 mmol L−1 ) of NaCl (Flowers and Colmer, 2008; Munns and Tester, 2008). More than 80% of land plants have symbiotic relationships with arbuscular mycorrhizal (AM) fungi (Brundrett, 2009). These soil fungi can promote plant growth under salt stress by supplying limiting nutrients and water to AM-colonized non-halophytes, including almost all grain crops (He and Nara, 2007; Sharifi et al., 2007; Daei et al., 2009). AM associations are also widely formed between AM fungi and halophytes, including Aster tripolium, Inula crithmoides, Plantago maritima, Salsola soda and Suaeda maritima (F¨ uzy et al., 2008; Evelin et al., 2009; Sonjak et al., 2009). The alleviation of salinity stress in AMcolonized halophytes is primarily through improved mineral content, water uptake, and osmolyte accumulation (Evelin et al., 2009). The biochemical mechanisms by which AM-colonized halophytic plants reduce or tolerate salt stress, however, are poorly understood. High salinity leads to an increased production of reactive oxygen species (ROS), including the superoxide radical (O− 2 ) and hydrogen peroxide (H2 O2 ) (Mittler, 2002). The excessive ROS can then lead to oxidative damage in the structures of enzymes and other macromolecules in plant cells. The induction of ROSscavenging antioxidant enzymes, including superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6), is the most common strategy used by plants to detoxify ROS synthesized during salt stress (Mittler, 2002; Evelin et al., 2009). SOD can convert O− 2 to H2 O2 (Wang et al., 2004), and CAT can catalyze H2 O2 to water and O2 and does not require a reducing substrate for its activity (Mittler, 2002). Up-regulation of antioxidant enzymes has been documented in AMcolonized non-halophytes. For example, during a 40day NaCl treatment, SOD activity under 0.5% and 1% NaCl was higher in the AM (Glomus mosseae)inoculated tomato seedlings than that in the non-AM controls, and CAT activity was also induced by this AM fungus (He et al., 2007). The roles of these enzymes in mycorrhizal plants, however, are poorly understood, especially under salt-stress conditions (He et al., 2007; Evelin et al., 2009). Moreover, little is known about the changes of SOD and CAT isoforms in AM plants under salt stress, except that the 32.0-kDa SOD isoform was significantly higher in non-mycorrhizal pea than in mycorrhizal (Pisum sativum) plants (Arines et al., 1994). Suaeda salsa L. is a leaf succulent C3 euhalophyte, whose high salt tolerance might be partly the result of

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its efficient antioxidative system (Wang et al., 2004; Pang et al., 2005). For instance, Mn-SOD and FeSOD activities in the leaves of S. salsa seedlings were significantly higher under NaCl stress conditions (100 mmol L−1 ) than those under non-NaCl stress conditions (Wang et al., 2004). Effects of AM fungi on leaf isoenzymes and activities of the antioxidant enzymes, however, have not been investigated in S. salsa. Given the potential of S. salsa to improve saline soils (Wang et al., 2004; Pang et al., 2005), the present study examined the effects of the AM fungus Glomus mosseae on the growth of S. salsa and on the activities and isoform patterns of the antioxidant enzymes SOD and CAT in S. salsa leaves under 400 mmol L−1 NaCl stress. Contents of malondialdehyde (MDA) were also determined to estimate the extent of free radical-induced lipid peroxidation, which could reflect the efficiency of the antioxidative system. MATERIALS AND METHODS Plants, mycorrhizal inoculum, medium

and plant growth

Seeds of Suaeda salsa L. were collected from Dongying City (37◦ 27 N, 118◦ 30 E), Shandong Province of China, and soaked for 5 h in tap water before germination. Inoculum of the mycorrhizal fungus G. mosseae, which included spores, hyphae, and colonized clover (Trifolium repens) root fragments, was obtained from a sand culture provided by the Institute of Mycorrhizal Biotechnology, Qingdao Agricultural University, Shandong Province of China. The plant growth medium, which was a mixture of sand and soil (3:1, w:w), was sterilized at 121 ◦ C for 2 h and then mixed with 100 g of autoclaved or nonautoclaved AM inoculum (1 700 inoculum potential unit) (Liu and Luo, 1994). The soil had a pH (H2 O) of 6.73, 8.2 g kg−1 of organic matter, and 69.04, 42.74, and 38.24 mg kg−1 of available nitrogen, phosphorus, and potassium, respectively. Experimental design and management An experiment was conducted with four treatments that included two levels of NaCl (0 and 400 mmol L−1 ) and two levels of G. mosseae (with and without). The NaCl level of 400 mmol L−1 was selected based on a preliminary experiment. Five replicates for each treatment were arranged in a completely randomized block design. Plants were grown in plastic pots (15 cm × 12 cm) in a greenhouse at light intensity of 800±100 μmol m−2 s−1 and relative humidity of 75%±5% for 15 h at

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27±3 ◦ C in day and 9 h at 17±3 ◦ C at night. The pots were irrigated weekly with P-free Hoagland solution or monthly with half-strength Hoagland solution for root mycorrhizal development. There were four uniform seedlings per pot. After cultivation of 60 d, seedlings were subjected to salt treatment. NaCl was dissolved in 1/2 strength Hoagland solution. NaCl concentrations were stepped up in 50 mmol L−1 per day until a concentration of 400 mmol L−1 on day 68. Control seedlings were irrigated daily with 1/2 strength Hoagland solution. These daily applications of 400 mmol L−1 NaCl and 1/2 strength Hoagland solution were continued until the experiment was terminated when the plants were 95 day-old. Plant harvest, mycorrhizal colonization, and enzyme analyses Number of leaves and branches, plant height, root length, and fresh and dry weights of shoots and roots were determined at the end of the experiment. Dry materials were oxidized at 550 ◦ C for 12 h; the residual was the inorganic dry weight, and the organic dry weight was the difference between the total and the inorganic dry weight. Colonization of roots by G. mosseae was assessed according to Kormanik and McGraw (1982). For SOD, CAT, and MDA analyses, leaves from the 3rd node and roots from each pot were sampled weekly during the period of NaCl stress (the last 35 days of the experiment). For each replicate pot, 1 g of leaves was ground to a fine powder in liquid nitrogen and then homogenized in 5 mL of extraction buffer (50 mmol L−1 phosphate buffered saline, pH 7.8, 0.1 mmol L−1 EDTA, 0.3% Triton X-100, 4% polyvinylpolypyrroidone). After centrifugation (10 500 × g, 4 ◦ C, 20 min), the supernatant was used for antioxidant enzyme analysis and isozyme electrophoresis. Protein concentration was determined with bovine serum albumin as the standard (Bradford, 1976). Activities of SOD and CAT in leaves were quantified according to Giannopolitis and Ries (1997) and Rao et al. (1997), respectively, and MDA content was quantified according to Draper and Hadley (1990). Separation and identification of SOD isoenzymes followed the methods of Fridovich (1986) and Wang et al. (2004) with modifications. Soluble proteins (20 μg) were loaded into each pocket of native polyacrylamide gel electrophoresis at 4 ◦ C with a 5% stacking and a 12% separating gel (w:v, acrylamide). Different SOD types were identified by adding inhibitors

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to the staining buffer containing 50 mmol L−1 phosphate buffered saline (pH 7.8), 10 mmol L−1 EDTA, 28 mmol L−1 tetramethylethylenediamine (TEMED), 30 μmol L−1 riboflavin, and 245 μmol L−1 nitro-blue tetrazolium chloride (NBT). KCN (3 mmol L−1 ) and H2 O2 (5 mmol L−1 ) were added to the buffer to inhibit Cu/Zn-SOD and Fe-SOD activities, respectively. Separation and identification of CAT isoenzymes were accomplished according to Woodbury et al. (1971) with modifications as follows. Soluble proteins (20 μg) were loaded into each pocket of native polyacrylamide gel electrophoresis at 4 ◦ C using a 5% stacking and a 7.5% separating gel (w:v, acrylamide). Gels were then reacted with a pre-cooled 0.3% H2 O2 solution and washed with pre-cooled distilled water. The washed gels were then put into a 2% FeCl3 and 2% K3 [Fe(CN)6 ] solution for visualization of bands. Bands of both SOD and CAT isoenzymes were scanned after staining and analyzed by the Bandleader 3.0 (Magnitec Ltd., Israel) and the Image Tool software (The University of Texas Health Science Center, San Antonio, USA). Statistical analyses Data were analyzed by analysis of variance (ANOVA). Differences in means were compared by the t-test and were considered significant at P ≤ 0.05. Costat (CoHort software, Berkeley, California, USA) and Microsoft Excel 2003 were used for statistical analyses. RESULTS AM root colonization AM colonization of roots was significantly lower for the plants treated with 400 mmol L−1 NaCl than that with 0 mmol L−1 NaCl (Table I). AM colonization decreased over time in the NaCl stress treatment but not in the control. Plant growth and leaf MDA content In general, the measured growth parameters (shoot height, dry weight, number of leaves and branches, root length, shoot organic and total plant dry weight) were significantly higher in the AM-inoculated seedlings than those in the non-AM-inoculated seedlings under either 0 or 400 mmol L−1 NaCl treatment (Table II). Water content, however, was unaffected by NaCl stress

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TABLE I Effect of salt treatment (0 or 400 mmol L−1 ) on colonization of Suaeda salsas roots by arbuscular mycorrhizal fungus (AMF) Glomus mosseae NaCl

mmol L 0 400

AMF colonization 0 da)

7d

14 d

21 d

35 d

44.7±0.4b) aAc) 44.9±0.4aA

44.8±1.0aA 40.6±1.0aB

% 45.4±0.9aA 33.8±1.0bB

46.0±1.0aA 29.0±0.7cB

46.8±1.2aA 19.7±0.7dB

−1

a)

Plants were 60 days old when the NaCl stress treatment was initiated. Means±standard errors (n = 5). c) Means followed by the same lowercase letter for a given NaCl level are not significantly different among sampling time at P ≤ 0.05; and those followed by the same uppercase letter for a given sampling time are not significantly different between 0 and 400 mmol L−1 NaCl levels at P ≤ 0.05. b)

TABLE II Effect of salt treatment (0 or 400 mmol L−1 ) and arbuscular mycorrhizal fungus (AMF) Glomus mosseae (without AMF or with AMF) on growth parameters of Suaeda salsa seedlings at the end of the experiment Parameter

0 mmol L−1 NaCl

400 mmol L−1 NaCl

Without AMF

With AMF

Fresh weight (g plant−1 ) Inorganic dry weight (g plant−1 ) Organic dry weight (g plant−1 ) Water content (g kg−1 ) Number of leaves Number of branches Shoot height (cm)

5.96±0.15a) bAb) 0.18±0.01bA 0.53±0.02bA 880.9±9.1aA 99.0±2.0bA 9.2±0.3aA 28.3±0.8bA

9.52±0.21aA 0.47±0.01aA 0.67±0.02aA 880.2±5.8aA 110.4±2.4aA 10.4±0.4aA 32.7±1.0aA

Fresh weight (g plant−1 ) Inorganic dry weight (g plant−1 ) Organic dry weight (g plant−1 ) Water content (g plant−1 ) Root length (cm)

3.04±0.21aA 0.03±0.00bA 1.29±0.12aA 565.8±7.5aA 11.1±0.4aA

Dry weight (g plant−1 )

2.03±0.15bA

Without AMF

With AMF

4.71±0.39bB 0.27±0.02bB 0.35±0.03bB 887.9±10.7aB 85.8±1.0bB 6.6±0.4bB 25.2±0.7bB

7.62±0.14aB 0.42±0.01aA 0.48±0.01aB 881.9±7.5aB 94.0±1.6aB 8.2±0.3aB 27.8±0.4aB

Shoot

Root 3.56±0.36aA 0.76±0.05bB 0.20±0.01aA 0.06±0.01bB 1.33±0.00aA 0.16±0.02bB 570.2±4.3aA 710.5±8.0bB 12.4±0.6aA 9.3±0.2bB Total plant 2.67±0.16aA 0.84±0.01bB

1.22±0.11aB 0.10±0.00aB 0.21±0.00aB 745.9±7.4aB 10.6±0.2aB 1.21±0.10aB

a)

Means±standard errors (n = 5). Means within a row followed by the same lowercase letter for a given NaCl level are not significantly different between the treatments with and without AMF at P ≤ 0.05; and those followed by the same uppercase letter under the same AMF treatment are not significantly different between 0 and 400 mmol L−1 NaCl levels at P ≤ 0.05. b)

or AM-inoculation. Lipid peroxidation is a useful indicator of cellular oxidative damage. MDA contents of leaves on all five sampling dates were similar between the treatments with and without AM fungi under 0 mmol L−1 NaCl stress (Fig. 1). In contrast, MDA contents in leaves were significantly higher in both the mycorrhizal and non-mycorrhizal plants under the 400 mmol L−1 NaCl treatment. Moreover, MDA contents in leaves on the

same sampling day were significantly lower in the mycorrhizal plants than in the non-mycorrhizal plants under the NaCl stress condition. SOD and CAT activities and their isoenzymes in leaves Under the non-saline condition and in the nonmycorrhizal plants, activities of SOD and CAT were unaffected by the sampling day, but SOD activity was

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Fig. 1 Effect of NaCl levels of 0 and 400 mmol L−1 (NaCl 0 and NaCl 400) and arbuscular mycorrhizal fungus (AMF) Glomus mosseae on malondialdehyde (MDA) content in the leaves of Suaeda salsa seedlings. The seedlings were grown for 60 d with and without AMF inoculation before the NaCl stress was applied. Bars with the same letter are not significantly different at P ≤ 0.05 (n = 5). Letters a, b, c, and d were used for the comparison among sampling time under the same NaCl level and AMF treatment; A and B for the comparison between 0 and 400 mmol L−1 NaCl levels on the same sampling time and with the same AMF treatment; and x and y for the comparison between the treatments with and without AMF on the same sampling time under the same NaCl level.

significantly higher in the mycorrhizal plants than that in the non-mycorrhizal plants except on the zero sampling day (Fig. 2). Also under the non-saline condition, CAT activity was significantly higher in the mycorrhizal plants than that in the non-mycorrhizal plants on the 14th and 21st sampling days (Fig. 2). In the 400 mmol L−1 NaCl treatments, SOD activity was significantly higher in the mycorrhizal plants than that in the non-mycorrhizal plants, while CAT activity was similar on the 7th and 35th days but was significantly higher on the 14th and 21st days in the mycorrhizal plants than that in the non-mycorrhizal plants. The reasons for changes in SOD and CAT activities in leaves were explored by analyzing their isoform patterns. After 14 days of NaCl stress (400 mmol L−1 NaCl), levels of isoenzymes of SOD (Fe-SOD, Cu/Zn-SOD1, and Cu/Zn-SOD2) and CAT (CAT1 and CAT2) in leaves were higher in the mycorrhizal plants than in the non-mycorrhizal plants (Fig. 3). This difference between mycorrhizal and non-mycorrhizal plants was also observed in the absence of NaCl stress (0 mmol L−1 NaCl). In addition, isoenzyme levels of SOD and CAT in leaves were higher in the plants under 400 mmol L−1 NaCl stress condition than under 0 mmol L−1 NaCl level. After 14 days of 400 mmol L−1 NaCl stress, the expression of Fe-SOD, Cu/ZnSOD1, and Cu/Zn-SOD2 in leaves (as calculated by the Bandleader 3.0) was 1.20, 1.19, and 1.07 times greater, respectively, in the mycorrhizal plants than in the non-mycorrhizal plants. Similarly, after 14 days

of 400 mmol L−1 NaCl stress, the expression of CAT1 and CAT2 in the leaves of mycorrhizal plants was 1.52 and 1.42 times greater than that in the leaves of nonmycorrhizal plants, respectively. DISCUSSION AM fungi help reduce salt stress in non-halophytes primarily through improved nutrient imbalance and water uptake (Evelin et al., 2009). The intensive mycelial networks of AM fungi could contribute to the improved nutrient uptake by halophytes (Wang et al., 2004; He et al., 2009). Previous results showed that AM fungi increased plant growth by reducing uptake of Na+ and Cl− (Tian et al., 2004; Al-Karaki, 2006) and by increasing uptake of P, K, Zn, Cu, and Fe (AlKaraki and Hammad, 2001; Al-Karaki, 2006; Daei et al., 2009). Consistent with previous studies, seedlings of the halophyte S. salsa grew better when colonized by G. mosseae under both 0 and 400 mmol L−1 NaCl stress (Table II). Our unpublished data indicated that the fungus reduced Na+ and Cl− accumulation in the shoots. In particular, in the 400 mmol L−1 NaCl treatment, mycorrhizal colonization increased plant dry weight by 44%; however, in the 0 mmol L−1 NaCl treatment, mycorrhizal colonization increased it only by 32%. Therefore, it is clear that colonization by G. mosseae improved salt tolerance of S. salsa. Whether mycorrhizal symbiosis also increased the uptake and translocation of P, K, Zn, Cu, and Fe to the shoots of

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Fig. 2 Effect of NaCl levels of 0 and 400 mmol L−1 (NaCl 0 and NaCl 400) and arbuscular mycorrhizal fungus (AMF) on superoxide dismutase (SOD) and catalase (CAT) activities in the leaves of Suaeda salsa seedlings. The seedlings were grown for 60 d with and without AMF inoculation before the NaCl stress was applied. Bars with the same letter are not significantly different at P ≤ 0.05 (n = 5). Letters a, b, c, and d were used for the comparison among sampling time under the same NaCl level and AMF treatment; A and B for the comparison between 0 and 400 mmol L−1 NaCl levels on the same sampling time and with the same AMF treatment; and x and y for the comparison between the treatments with and without AMF on the same sampling time under the same NaCl level.

the halophyte S. salsa under salinity requires further study. Surprisingly, shoot water content was unaffected by inoculation or non-inoculation with G. mosseae regardless of salt treatment (Table II). No data had been published regarding the effects of AM fungi on water content of halophyte plants under salt stress. Our earlier results showed that salinity significantly enhanced the leaf succulence of S. salsa seedlings via upregulation of aquaporins (Qi et al., 2009). Additional research is needed to determine whether halophytic plants, when colonized by AM fungi, have mechanism(s) for coping with water balance under salt stress. The ability of plants to tolerate salt stress is also determined by the efficiency of antioxidative enzymes (Wang et al., 2004). However, the effects of AM fungi on the synthesis and activities of antioxidant enzymes in halophytes have been seldom studied. We compared the effects of a common AM fungus, G. mosseae, un-

der salt stress and non-salt stress conditions, on SOD and CAT activities and on the expression of SOD and CAT isoenzymes. These enzymes are involved in the scavenging of ROS in the C3 euhalophyte S. salsa. Whether or not the plants were treated with NaCl, G. mosseae significantly increased the activity of SOD (Fig. 2), which is consistent with the results reported for Olea europaea, Retama sphaerocarpa, soybean and tomato (Alguacil et al., 2003; Ghorbanli et al., 2004; He et al., 2007). Moreover, the levels of SOD isoforms (Fe-SOD, Cu/Zn-SOD1 and Cu/Zn-SOD2) were significantly increased by G. mosseae, although no differences were detected among the types of SOD isoforms (Fig. 3). Compared with the non-mycorrhizal plants, mycorrhizal S. salsa had higher expression of Fe-SOD, Cu/Zn-SOD1 and Cu/Zn-SOD2. These SOD isoenzymes scavenge ROS induced by salt stress and thus reduce the damage of ROS to the plants (Mittler, 2002).

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determined from the AM fungi Gigaspora margarita (Lanfranco et al., 2005), Gigaspora rosean, and four Glomus species (Corradi et al., 2009). One possibility is that mycorrhizal symbioses enhance specific phytohormones throughout the plant (Barker and Tagu, 2000). Another possibility is that mycorrhizal symbioses improve nutrient balance in shoots under salt stress (Al-Karaki and Hammad, 2001; Al-Karaki, 2006; Daei et al., 2009). The changes of phytohormones in plant and nutrients in shoots could then regulate the antioxidative enzymes. Further research is needed to elucidate the molecular mechanisms of how AM fungi increase antioxidative enzymes and reduce salt stress in halophytes. Fig. 3 Effect of NaCl levels of 0 and 400 mmol L−1 (NaCl 0 and NaCl 400) and arbuscular mycorrhizal fungus (AMF) on patterns of superoxide dismutase (SOD) isoenzymes (a) and catalase (CAT) isoenzymes (b) in the leaves of Suaeda salsa seedlings. The seedlings were harvested 14 days after the initiation of the salt stress. Twenty micrograms of protein per pocket was loaded and separated on the native polyacrylamide electrophoresis.

This was evidenced by the decreased content of MDA in the mycorrhizal S. salsa seedlings under NaCl stress (Fig. 1); MDA is a specific product of lipid peroxidation induced by ROS. These results for the euhalophyte S. salsa are consistent with those observed for soybean (Glycine max) (Porcel and Ruiz-Lozano, 2004). Significantly higher CAT activity in leaves of S. salsa seedlings colonized by G. mosseae under salt stress was also consistent with the results obtained with O. europaea, R. sphaerocarpa, and soybean (Alguacil et al., 2003; Ghorbanli et al., 2004), but not with tomato (He et al., 2007). To the best of our knowledge, our study is the first to document a higher level of CAT isoforms (CAT1 and CAT2) in leaves of mycorrhizal S. salsa seedlings under salt stress (Fig. 3). The function of CAT is to detoxify H2 O2 . Higher CAT activity and higher levels of CAT1 and CAT2 isoforms could thus help plants to reduce oxidative damage under salt stress. On the other hand, AM fungi could also enhance the activities of other antioxidative enzymes, such as ascorbate peroxidase (APX) and peroxidase (POD), as observed for mycorrhizal O. europaea, R. sphaerocarpa, tomato, and Ziziphus xylopyrus under salt stress (Mathur and Vyas, 1996; Alguacil et al., 2003; He et al., 2007). However, little is known about how AM fungi, which colonize roots, can enhance antioxidative enzymes in plant shoots under salinity, although the sequence of the Cu/Zn-SOD gene has been

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