Effects of salt stress on ion content, antioxidant enzymes and protein profile in different tissues of Broussonetia papyrifera

South African Journal of Botany 85 (2013) 1–9

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Effects of salt stress on ion content, antioxidant enzymes and protein profile in different tissues of Broussonetia papyrifera Min Zhang a, Yanming Fang b, Yonghua Ji a,⁎, Zeping Jiang a,⁎⁎, Lei Wang a a b

Jiangsu Academy of Forestry, Dongshanqiao, Jiangning, Nanjing, Jiangsu, 211153, China College of Forest Resources and Environment, Nanjing Forestry University, 159 Longpan Road, 210037, China

a r t i c l e

i n f o

Article history: Received 17 May 2012 Received in revised form 11 November 2012 Accepted 14 November 2012 Available online 23 December 2012 Edited by P Berjak Keywords: Broussonetia papyrifera Antioxidant enzymes Protein profile NaCl stress

a b s t r a c t Although some plant responses to salinity have been characterized, the precise mechanisms by which salt stress damages plants are still poorly understood especially in woody plants. In the present study, the physiological and biochemical responses of Broussonetia papyrifera, a tree species of the family, Moraceae, to salinity were studied. In vitro-produced plantlets of B. papyrifera were treated with varying levels of NaCl (0, 50, 100 and 150 mM) in hydroponic culture. Changes in ion contents, accumulation of H2O2, as well as the activities and isoform profiles of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in the leaves, stems and roots were investigated. Under salt stress, there was higher Na+ accumulation in roots than in stems and leaves, and Ca 2+, Mg2+ and P 3+ content, as well as K+/Na+ ratio were affected. NaCl treatment induced an increase in H2O2 contents in the tissues of B. papyrifera. The work demonstrated that activities of antioxidant defense enzymes changed in parallel with the increased H2O2 and salinity appeared to be associated with differential regulation of distinct SOD and POD isoenzymes. Moreover, SDS-PAGE analysis of total proteins extracted from leaves and roots of control and NaCl-treated plantlets revealed that in the leaves salt stress was associated with decrease or disappearance of some protein bands, and induction of a new protein band after exposure to 100 and 150 mM NaCl. In contrast, NaCl stress had little effect on the protein pattern in the roots. In summary, these findings may provide insight into the mechanisms of the response of woody plants to salt stress. © 2012 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Salinity is one of the major abiotic stresses, which has a major impact on plant production and productivity (Zhu, 2001). Excess amount of salt in the soil adversely affects plant growth and development (Sairam and Tyagi, 2004). High salt level causes ion imbalance and hyperosmotic stress in plants (Zhu, 2001), which has toxic effects on numerous biochemical processes. Salinity affects plants in many different ways including osmotic effects, which resulted from increased osmotic potential of the soil solution that makes the water in the soil less available for plants (Eraslan et al., 2007), specific-ion toxicity and secondary stresses such as oxidative damage (Zhu, 2001). One of the biochemical changes occurring when plants are subjected to salt stress is the accumulation of reactive oxygen species (ROS) such as superoxide (O2−), hydrogen peroxide (H2O2), and Abbreviations: APX, ascorbate peroxidase; BSA, bovine serum albumin; CAT, catalase; DHAR, dehydroascorbate reductase; DTT, dithiothreitol; GPX, guaiacol peroxidase; MDHAR, monodehydroascorbate reductase; PAGE, polyacrylamide gel electrophoresis; POD, peroxidase; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate, SOD, superoxide dismutase; TEMED, N,N,N′,N′-tetramethylethylenediamine. ⁎ Corresponding author. Tel.: +86 25 52741622; fax: +86 25 52741620. ⁎⁎ Corresponding author. Tel.: +86 25 52745600. E-mail addresses: [email protected] (Y. Ji), [email protected] (Z. Jiang).

hydroxyl radicals (OH•) (Van Breusegem et al., 2001). ROS can seriously disrupt normal metabolism through oxidative damage to lipids, protein and nucleic acids (Meloni et al., 2003), and damage membrane function (Gόmez et al., 2004). Plants have evolved a series of antioxidant systems that protect them from these potential cytotoxic effects (Rahnama and Ebrahimzadeh, 2005), such as the system that reacts with active forms of oxygen and keeps them at a low level (e.g. superoxide dismutase, catalase and peroxidases), and the system that regenerates oxidized antioxidants (e.g. glutathione reductase and ascorbate peroxidase) (Smirnoff, 1993). The existence of multiple molecular forms of antioxidant enzymes, and any changes they may undergo in response to various environmental signals imply potential roles for these isozymes in the detoxification of ROS (Pinhero et al., 1997). The isozymes could be used as a biochemical marker to study the tolerance of plant to stress (Ying et al., 2006). Many studies have been carried out to study the isozymes of plant correlated with their tolerance to stress by isozyme analysis. The altered antioxidant enzyme activities have been described in plant species under abiotic stresses including salinity (Kim et al., 2005; Elkahoui et al., 2005; Jebara et al., 2005; Rahnama and Ebrahimzadeh, 2005). In barley roots under salt stress, the activities of antioxidant enzymes increased significantly, among which CAT activity increased most drastically. And the significant increase in the

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activities of the antioxidant enzymes in NaCl-stressed barley root was highly correlated with the increased expression of the constitutive isoforms as well as the induced ones (Kim et al., 2005). Study on antioxidant enzyme expression in Catharanthus roseus suspension cells in response to salt stress indicated that the antioxidant enzyme activity and isoenzymes were influenced by salt stress (Elkahoui et al., 2005). The same results were also found in common bean (Phaseolus vulgaris) nodules (Jebara et al., 2005) and potato seedlings (Rahnama and Ebrahimzadeh, 2005) under salt stress. Protein synthesis can be affected by environmental stresses including water (Bewley et al., 1983) and salt stress (Hurkman and Tanaka, 1987; Sousa et al., 2003). Several salt-induced proteins have been identified (Singh et al., 1987; Fisher et al., 1994; Ali et al., 1999; Sousa et al., 2003), which belong to two distinct groups: salt stress proteins that accumulate only due to salt stress, and stress associated proteins, which accumulate in response to various environmental stresses including heat, cold, drought, waterlogging, etc. (Ashraf and Harris, 2004). Proteins accumulated in plants grown under saline conditions may provide a storage form of nitrogen which could be reutilized when stress is over (Singh et al., 1987), and they may play a role in osmotic adjustment (Qasim et al., 2003). These proteins may be synthesized de novo in response to salt stress or may be present constitutively at low concentration and increase when plants are exposed to salt stress (Pareek et al., 1997). Broussonetia papyrifera, family Moraceae, is a large shrub or small tree. The bark of B. papyrifera is a source of fiber for making paper and cloth and the leaves, fruit and bark have a variety of traditional medicinal uses. Due to its fast growth this tree can rapidly colonize forest clearings and abandoned farmland. Broussonetia papyrifera is tolerant to drought and resistant to salt stress, making it a suitable woody plant for saline environments (Yang et al., 2009). To gain insight into the mechanism underlying salt stress on woody plants, changes in antioxidant enzyme activities and isoenzymes of SOD, POD and CAT, as well as protein profiles in different tissues of B. papyrifera in response to NaCl treatment were investigated in the present study. And the tissue-specific responses of the antioxidant enzymes and protein expression to salt stress were analyzed. 2. Materials and methods

for 48 h and the dry weight was measured. The oven-dried ground material (0.5 g) was digested with 8 mL of sulfuric acid-hydrogen peroxide mixture. The concentrations of Na +, K +, Ca 2+, Mg 2+ and P 3+ in the digests were determined with an inductively coupled plasma-atomic emission spectroscopy (ICP-AES, Perkin Elmer Optimal 2100-DV). 2.3. Determination of H2O2 The H2O2 content was colorimetrically measured as described by Mukherjee and Choudhuri (1983). 2.4. Antioxidant enzymes activity assay The activities of SOD, POD and CAT were determined spectrophotometrically with a Beckman DU 800 UV/VIS spectrophotometer. SOD activity was measured according to the protocol of Yu and Rengel (1999). One unit of SOD activity was defined as the amount of enzyme that causes 50% inhibition of the photochemical reduction of NBT, and SOD specific activity was expressed as units per mg protein (Rodríguez et al., 2007). Specific POD activity was determined by measuring the oxidation of benzidine at 530 nm according to the previous report (Rahnama and Ebrahimzadeh, 2005). Total CAT activity was analyzed by measuring the consumption of H2O2 at 240 nm according to the method of Beers and Sizer (1952), and enzyme activity expressed as U mg−1 protein. 2.5. Enzyme extraction Enzyme extractions were carried out at 4 °C according to the previously reported procedures (Pereira et al., 2002). Plant tissues were frozen in liquid nitrogen and ground with an ice-cold pestle and mortar, and then extracted in 100 mM potassium phosphate buffer (pH 7.5) containing 1 mM EDTA, 3 mM DTT and 5% (w/v) insoluble PVP in the ratio of 1:3 (w/v). The homogenate was passed through four layers of cheesecloth and then centrifuged at 10,000 ×g for 30 min. The supernatant was collected and stored in small aliquots at − 80 °C for SOD, POD and CAT analysis. Protein was determined by the method of Bradford (1976) using BSA as a standard.

2.1. Plant culture and salt treatment 2.6. Native gel electrophoresis and enzyme activity staining B. papyrifera shoot segments with axillary buds were used as explants. The explants were transferred to the shoot multiplication media composed of Murashige and Skoog (MS) basal salts, 1 mg L −1 6-BA, 0.1 mg L −1 IBA, 30 g L −1 sucrose and 6.5 g L−1 agar, pH 5.8, and the cultures maintained at 25± 2 °C in a 16 h light and 8 h dark cycle, with an irradiance of 1500 lux. For root induction, half-strength MS medium supplemented with 0.5 mg L −1 IBA, 0.3 mg L −1 NAA, 30 g L −1 sucrose and 6.5 g L −1 agar, pH 5.8 was utilized, and the cultures were maintained as described above. Fifty in vitro regenerated B. papyrifera rooted plantlets of uniform size were planted into plastic pots filled with 500 mL of half-strength MS solution. A plastic sheet was placed over each pot to prevent evaporation. Plants were maintained in a growth chamber under controlled conditions. The nutrient solution was changed every other day. For salt treatment, four NaCl levels, 0 mM (with no NaCl added), 50 mM, 100 mM and 150 mM NaCl were established with three replicates. After five days of treatment the leaves, stems and roots were harvested. 2.2. Determination of ion contents The harvested plants were quickly rinsed in deionized water to remove ions from the free space and the excessive water was dried with a paper towel. And the fresh weights of the roots, stems and leaves were harvested. Afterwards, the samples were oven-dried at 60 °C

The isozymes of antioxidant enzymes were visualized by nondenaturing polyacrylamide gel electrophoresis (native-PAGE) on a Mini-Protean Ш slab gel cell (Bio-Rad, Hercules, CA). Equal amounts of protein were loaded onto each lane. And the gels were scanned with a ChemiDoc XRS imaging system (Bio-Rad) after the photochemical reaction. Isozymes of SOD were separated on 10% polyacrylamide gel and stained according to Fornazier et al. (2002) with minor modification. After electrophoresis, the gels were washed extensively with distilled water and soaked in 50 mM potassium phosphate buffer (pH 7.8) containing 1 mM EDTA, 0.05 mM riboflavin, 0.1 mM nitroblue tretazolium and 0.3% (v/v) N,N,N′,N′-tetramethylethylenediamine (TEMED), and incubated in the dark for 30 min at room temperature. After incubation, gels were exposed to the light for about 50 min. And then the gels were rinsed with distilled water and the colorless SOD bands were visible against a purple background. POD isoenzymes were visualized by immersing the gels in 0.2 M acetate buffer (pH 4.8) containing 0.3% H2O2 and 4% benzidine in 50% methanol at room temperature until the brown color appeared (Van Loon, 1971). For the staining of CAT, the method of Woodbury et al. (1971) was adopted. Briefly, the gels were rinsed with distilled water and then incubated in 0.3% H2O2 for 10 min. After being washed with distilled

M. Zhang et al. / South African Journal of Botany 85 (2013) 1–9

water the gels were stained with a mixture of 2% potassium ferricyanide and 2% ferric chloride. 2.7. Protein extraction and SDS-PAGE Total protein was extracted by the modified acetone precipitation method (Gu et al., 1999). Plant materials (1 g) were homogenized in the protein extraction buffer (62.5 mM Tris–HCl, pH 6.8, 0.5% SDS, 10% glycerol and 5% β-mercaptoethanol) containing 10% PVP. The homogenate was then centrifuged at 12,000 ×g for 20 min. Proteins were precipitated in cold acetone at −20 °C for 1 h, followed by centrifugation at 12,000 ×g for 10 min at 4 °C. The pellets were air dried and the extracted protein was dissolved in solubilizing buffer comprised of 62.5 mM Tris–HCl, pH 6.8, 10% glycerol, 5% β-mercaptoethanol and 2 mM PMSF. After centrifugation (12,000 ×g, 10 min at 4 °C), the supernatant was collected for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Electrophoresis was carried out on 12% polyacrylamide gels. Gels were run for 30 min at 50 V and 80 min at 150 V. After completion of electrophoresis, the gels were stained with coomassie brilliant blue R-250. Images were acquired using the ChemiDoc XRS system (Bio-Rad) and analyzed with Quantity One-1D software. The molecular weights of the protein bands were estimated using molecular weight markers (Fermentas Inc., Glen Burnie, Maryland). 2.8. Data analysis Data were evaluated by one-way ANOVA using SPSS 13.0 software. Significant differences among treatments were calculated by Duncan's multiple range tests (P = 0.05). 3. Results 3.1. Effect of NaCl stress on the growth of B. papyrifera The effect of NaCl on plant growth was assessed by measuring the fresh and dry weight of the leaf, stem and root of B. papyrifera. The results showed that 50 and 100 mM NaCl significantly elevated the fresh weight of the leaf and stem (Table 1). In contrast, the fresh weight of the root was not affected by 50 and 100 mM NaCl treatment. As for the dry weight, only that of the stem was significantly enhanced by 100 mM NaCl. Moreover, 150 mM NaCl brought about significant reductions in fresh and dry weight of the leaf and root. However, only the fresh weight of the stem was decreased by 150 mM NaCl. 3.2. Changes in ion contents and K +/Na + ratio in response to NaCl stress As shown in Table 2, there was a positive relationship for Na + content in tissues with the increase in NaCl concentration. In the presence of 50–150 mM NaCl, all treatments induced a significant increase in Na + content, such an effect being the most pronounced for the roots in which 150 mM NaCl led to 50.4 folds increase in Na +

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concentration. In the roots, K + content decreased with the increase in NaCl concentration. However, K + content showed no appreciable changes in the stem. The uptake and partitioning of Ca 2+ varied among different tissues. A negative relationship was obtained for Ca 2 + content with salt concentration in the roots. The concentration of Ca 2+ significantly declined with increase in salt stress, whereas there were no obvious changes in Ca 2 + content in the stem and leaf. In addition, NaCl stress had almost no impact on Mg 2+ and P 3+ in B. papyrifera, except for the decline of P 3+ concentration in the leaf. Moreover, there was a negative relationship between K +/ Na + ratio and increase in salt stress. The K +/Na + ratio significantly decreased in tissues with increase in salinity. 3.3. Elevation of H2O2 levels in NaCl-treated plants The levels of H2O2 in different tissues of NaCl-treated plants were determined. In our study, NaCl treatment increased H2O2 accumulation in the leaf, stem and root of B. papyrifera (Fig. 1). And the accumulation of H2O2 was obvious both in the stem and leaf. The content of H2O2 in the stem in 150 mM NaCl treatment was 4.3 fold higher than that in the control. 3.4. Alteration of antioxidant enzymes activity under NaCl stress Since NaCl treatment induced an increase in the amount of H2O2 in the tissues of B. papyrifera. We then examined changes in the activity of the antioxidant enzymes including SOD, POD and CAT in response to NaCl stress. The results revealed that NaCl stress had different effects on the activity of these three antioxidant enzymes. There were no marked differences in leaf SOD activity under different NaCl concentrations (Fig. 2A). In addition, SOD activity of the stem was higher in salt treated plants than that in the control. Conversely, SOD activity of the root showed a continuous decrease, declining 40.3% at 150 mM NaCl as compared with control. POD activity showed no significant change at 50 mM NaCl in the leaf, whereas it decreased at 100 and 150 mM NaCl (Fig. 2B). In contrast, POD activity decreased dramatically at 50 mM NaCl in the stem, while 100 and 150 mM NaCl treatment did not induce significant differences in POD activity in comparison with the control. However, in the root 100 and 150 mM NaCl treatments caused 25.7% and 64.8% decrease in POD activity in comparison with the control plants. There was a decline in CAT activity while increasing the magnitude of NaCl stress in the leaf, and CAT activity remained only 15.8% of the activity at 100 mM NaCl in comparison with the control (Fig. 2C). Similarly, 100 mM NaCl treatment also decreased CAT activity in the stem, whereas a significant increase in the activity of CAT was observed at 150 mM NaCl. Different from the leaf and stem, NaCl treatment had no significant impact on CAT activity in the root. 3.5. Isozyme patterns of the antioxidant enzymes under NaCl stress Since we observed alterations in the activity of antioxidant enzymes under NaCl stress, we further explored the impact of NaCl

Table 1 Effect of NaCl stress on fresh weight (FW) and dry weight (DW) of the leaf, stem and root of Broussonetia papyrifera. NaCl (mM)

Leaf

Stem

FW (mg plant−1) 0 50 100 150

133.8 ± 4.4 156.5 ± 4.8 152.6 ± 5.5 101.3 ± 2.4

b a a c

DW (mg plant−1) 19.6 ± 1.2 25.2 ± 1.8 26.1 ± 3.1 11.9 ± 0.8

a a a b

Root

FW (mg plant−1) 149.2 ± 6.4 168.3 ± 3.8 172.7 ± 4.8 119.8 ± 2.4

b a a c

DW (mg plant−1) 12.2 ± 0.9 15.7 ± 1.3 17.4 ± 2.1 8.9 ± 1.0

bc ac a b

FW (mg plant−1) 75.1 ± 3.3 85.6 ± 3.1 83.6 ± 2.5 42.7 ± 4.0

a a a b

DW (mg plant−1) 7.3 ± 0.6 8.5 ± 0.6 9.1 ± 0.8 4.6 ± 0.3

a a a b

Values were mean ± SEM of three independent experiments. Different letters next to values within each column indicated statistically different means at the P b 0.05 level.

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Table 2 Effect of NaCl stress on ion contents of Broussonetia papyrifera. Tissue

NaCl concentration (mM)

Na+ (mg/g DW)

Root

0 50 100 150 0 50 100 150 0 50 100 150

1.02 ± 0.03 20.36 ± 0.17 30.69 ± 0.21 51.45 ± 0.45 14.59 ± 0.36 15.78 ± 0.89 22.45 ± 1.95 29.89 ± 0.70 1.94 ± 0.30 4.39 ± 0.09 8.60 ± 0.22 11.70 ± 0.15

Stem

Leaf

K+ (mg/g DW) d c b a c c b a d c b a

46.88 ± 0.30 39.04 ± 0.89 46.98 ± 0.38 34.86 ± 0.19 57.68 ± 0.24 54.12 ± 1.33 55.18 ± 3.46 56.09 ± 1.05 53.71 ± 0.94 51.11 ± 0.34 47.66 ± 0.42 52.40 ± 0.54

Ca2+ (mg/g DW) a b a c a a a a a b c ab

6.48 ± 0.08 6.91 ± 0.13 4.45 ± 0.06 4.97 ± 0.02 7.54 ± 0.09 7.38 ± 0.26 7.89 ± 0.41 6.55 ± 0.10 12.23 ± 0.32 11.24 ± 0.04 11.76 ± 0.18 11.96 ± 0.12

Mg2+ (mg/g DW) b a d c a a a a a b ab a

2.94 ± 0.02 2.76 ± 0.06 2.60 ± 0.01 2.31 ± 0.01 1.32 ± 0.05 1.30 ± 0.04 1.35 ± 0.08 1.15 ± 0.01 2.62 ± 0.05 2.48 ± 0.02 2.45 ± 0.02 2.56 ± 0.01

P3+ (mg/g DW) a b c d a a a a a b b b

3.47 ± 0.04 3.86 ± 0.32 3.45 ± 0.03 3.18 ± 0.08 1.97 ± 0.03 2.06 ± 0.19 2.06 ± 0.23 2.38 ± 0.18 2.09 ± 0.11 2.08 ± 0.09 1.74 ± 0.13 1.54 ± 0.02

K+/Na+ ab a ab b a a a a a ab c cd

46.24 ± 1.13 1.92 ± 0.05 1.53 ± 0.01 0.68 ± 0.01 3.96 ± 0.05 3.43 ± 0.03 2.59 ± 0.02 1.92 ± 0.02 29.21 ± 5.04 11.66 ± 0.25 5.55 ± 0.10 4.48 ± 0.05

a b b b a b c d a b b b

Data presented were mean ± SEM for three replicates. Different letters within each column indicated significant difference among treatments at the P b 0.05 level.

on the isozyme profiles. Nondenaturing PAGE coupled with activity localization showed four SOD isozymes in the leaves (Fig. 3A, B). And SOD2 and SOD3 were the predominant isozymes. The results also indicated that none of the four isozymes were strikingly affected by salt stress. Examination of SOD isoenzyme profiles in the stems revealed five isoforms (SOD1-5) (Fig. 3C, D), and the activity of SOD1 and SOD5 declined with increase in NaCl concentration (Supplementary Fig. 1A). Whereas, in the roots of the control only some faint bands were detected (Fig. 3E, F). In 50 mM NaCl an isozyme was induced and another isozyme SOD2 appeared in 50, 100 and 150 mM NaCl-treated plantlets. Meanwhile, SOD3 and SOD4 were activated under 150 mM NaCl (Supplementary Fig. 1B). In the untreated control leaves of B. papyrifera, three isozymes of POD (POD1, POD2 and POD3) were observed following staining of PAGE gels (Fig. 4A, B). When the plantlets were exposed to 50 mM NaCl, POD1 and POD3 disappeared. However, POD1 and POD3 reappeared when the plantlets were exposed to NaCl at 100 and 150 mM, and a clear increase in the activity of POD3 was detected at 150 mM NaCl treatment (Fig. 4A, B and Supplementary Fig. 2A). In contrast to the leaves, more bands were visualized in the stems in response to NaCl treatment (Fig. 4C, D). In the control a total of four bands were revealed. However, shifts in the isozyme bands were observed after NaCl treatments. In stems of the plantlets treated with 50 mM NaCl, five bands were detected and among which three bands were induced by NaCl. Moreover, POD active staining exhibited a similar isoenzyme pattern in 100 and 150 mM NaCl treatment. As compared with the isozyme band pattern in the stem, there were fewer bands presented in the roots (Fig. 4E, F). There were only two bands present in the control and four bands in NaCl-treated plantlets (POD1-4). Two bands appeared in response to NaCl treatment, which were POD2 and POD4. Moreover, the activities of POD1 and POD3 were elevated by 150 mM NaCl (Supplementary Fig. 2B).

Fig. 1. Effects of NaCl on H2O2 accumulation in B. papyrifera seedlings. The values were mean ± SEM for three replicates. Different letters on each bar represent significant difference at the P = 0.05 level.

As shown in Fig. 5, only one prominent CAT band was detected in the leaves, stems and roots of B. papyrifera. Quantification of the band intensities showed that there was a decline in CAT activity in the leaves (Fig. 5A) and roots (Fig. 5C) after NaCl treatment. As compared to the leaves and roots, 50 mM NaCl treatments had no effect on stem

Fig. 2. Changes in SOD (A), POD (B) and CAT (C) activities in the crude extracts obtained from leaves, stems and roots of B. papyrifera in varying concentrations of NaCl (0, 50, 100 and 150 mM). Data represented mean ± SEM of three independent experiments. Bars with the same letters are not significantly different at the P = 0.05 level based on Duncan's multiple range tests.

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Fig. 3. Pattern of SOD activity bands in leaves (A), stems (C) and roots (E) of B. papyrifera growing under different concentrations of NaCl. The extracts were separated by native PAGE and stained for SOD activity. Images were captured with a ChemiDoc XRS imaging system. Densitometric scans of SOD isoenzymes from leaves (B), stems (D) and roots (F) were performed by Quantity One 1-D software. The numbers indicate different isoformic bands.

CAT activity, whereas 100 mM NaCl caused a decline in CAT activity (Fig. 5B). 3.6. Effects of NaCl stress on soluble protein content Salt stress had no significant effect on the concentration of soluble proteins either in the leaves or in the roots at 50 mM NaCl (Fig. 6). In contrast, 100 mM NaCl treatment induced a significant increase in soluble proteins in the leaves, while only little change was detected

in the roots. However, the concentration of the soluble proteins recovered to the control level when NaCl concentration was elevated to 150 mM in the leaves, while no significant change was detected in the roots. 3.7. NaCl affected the protein profiles of leaves and roots of B. papyrifera Proteins were extracted from the leaves and roots of control and NaCl-treated plants and analyzed by SDS-PAGE. Protein profiles of

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M. Zhang et al. / South African Journal of Botany 85 (2013) 1–9

Fig. 4. Isoenzyme pattern of POD in B. papyrifera seedlings treated with various concentrations of NaCl (0–150 mM). Proteins from leaves (A), stems (C) and roots (E) were electrophoresed by native PAGE and the isoenzymes of POD were visualized as described in Section 2. The isoenzymes resolved are denoted by different numbers. Densitometric scans of POD isoenzymes on the profiles from leaves, stems and roots are shown in Figs B, D and F, respectively.

the leaves and roots were demonstrated in Fig. 7. It showed that NaCl treatment induced alterations in protein expression pattern and level in the leaves (Fig. 7A). 100 mM and 150 mM NaCl induced a new protein band in the leaf and densitometric scanning of the gel revealed that the intensity of this new band increased with increasing NaCl concentration (Fig. 7B). However, the protein band with a molecular weight of 86 kDa disappeared at 100 mM NaCl and the 60 kDa protein band diminished at 50 mM NaCl. Moreover, we observed that the level of 72 kDa and 41 kDa protein decreased in NaCl-treated plantlets, whereas, the expression of 16 kDa protein increased in response to NaCl treatment. In contrast to the leaves, NaCl stress had

little effect on the protein pattern in the roots. As shown in Fig. 7C and D, the protein bands of molecular weight 70, 52 and 46 kDa decreased as a result of NaCl treatment. But the 17 kDa protein band was elevated by NaCl treatment. 4. Discussion Soil salinity is a major abiotic stress, which can decrease water potential and induce nutrient imbalances in plants, and these adversely affect plant growth (Mansour and Salama, 2004; Chinnusamy et al., 2005; Genc et al., 2007). Therefore, understanding the molecular

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Fig. 5. Activity staining for CAT of extracts of B. papyrifera leaves (A), stems (B) and roots (C) following native PAGE. Only one isozyme of CAT was observed in different B. papyrifera tissues. The relative intensity of CAT isozyme under different NaCl treatment was analyzed by Quantity One 1-D software.

basis of salt-stress tolerance mechanisms are essential for breeding and genetic engineering of salt tolerance in plants (Chinnusamy et al., 2005). Despite intensive research over the past several decades, which has improved our understanding of plants' response to salinity, there have been few studies on the mechanisms of the adaptation of tree species to salinity. In the present study, we conducted experiments to test the response of B. papyrifera elicited by salinity. The challenge by NaCl resulted in imbalance of ions in the plants depicted by increased Na + concentration and reduced K + and Ca 2+ contents. Moreover, it demonstrated that the antioxidant enzyme activities and isoenzymes as well as protein profiles in the leaves, stems and roots of B. papyrifera had different responses to NaCl treatment. High salt concentration in the external solution causes ion imbalance or disturbances in ion homeostasis (Parida and Das, 2005). Previous studies have revealed that there exists competition between Na + and K +, which lead to a reduced level of internal K + at high external NaCl concentration (Rus et al., 2004; Kaya et al., 2007). The influenced uptake of K + by NaCl leading to K + deficiency, and it can enhance the ratio of Na +/K + that reduce plant growth and causes ionic toxicity (Cuin et al., 2003; Kaya et al., 2007). Our study revealed that increased NaCl concentration induced a significant decrease in K + uptake and a high Na +/K +. It was considered that Na + competes with the uptake of K + was due to the chemical similarities between both ions (Borsani et al., 2003). In addition, it has been reported that NaCl treatment induced decrease in Ca 2+ and Mg 2+ levels in the plants (Khan et al., 2000; Khan, 2001). In the present

Fig. 6. Effects of NaCl on soluble protein contents in the leaves and roots of B. papyrifera seedlings. Data were presented as mean± SEM for three replicates. Letters on each bar represent significant difference at the P = 0.05 level.

study, we also observed a decrease in Ca 2+ content in the roots. In contrast to the previous report NaCl treatment had little effect on Mg 2+ level. It may suggest that different plant species have different response to salt stress. Plants exposed to salinity were prone to oxidative stress because of the formation of ROS such as O2−, H2O2 and OH (Van Breusegem et al., 2001; Parida and Das, 2005). These ROS can affect the integrity of cellular membranes, enzymes activities and the plant photosynthetic apparatus (Serrano et al., 1999; Jithesh et al., 2006). Plant cells contain an array of antioxidant enzymes that scavenge or prevent the formation of the aggressive ROS, which protect cells from oxidative damage (Matamoros et al., 2003). There have been reports of increasing activity of antioxidative enzymes under salt stress. Activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) were reported to increase in Pea (Pisum sativum L.). Evidence showed that salt treatment enhanced the activity of APX, guaiacol peroxidase (GPX) and SOD, and it induced the decrease of CAT activity in the leaves of B. parviflora (Parida et al., 2004a). SOD, POD and CAT are usually considered as the key components of antioxidant defense of the plants (Xue et al., 2008). In the present study, the changes of SOD, POD and CAT enzyme activities as well as the expression of isozymes in B. papyrifera were examined. In the leaves, 100 and 150 mM NaC1 treatment decreased POD and CAT activity with no impact on SOD activity. In contrast, SOD, POD and CAT activities in the stems were significantly elevated under high NaCl concentration. Whereas, in the roots, SOD and POD activities decreased and CAT activity had little change under NaCl stress. It indicated that salt-stressed B. papyrifera plants exhibited specific responses of SOD, POD and CAT activities. The utilization of multiple isoforms of antioxidant enzymes is one of the mechanisms involved in preventing the damage caused by ROS (Kim et al., 2005). Previous studies have indicated that NaCl stress can either stimulate or inhibit the expression of the isoforms of several antioxidant enzymes. The increase of constitutive isoforms of SOD, CAT, POX and GR was observed in NaCl-treated barley root and shoot. Additionally, the induction of new isoforms was also found in CAT and GR in the shoot, and APX in the root (Kim et al., 2005). In potato seedlings, new POD and SOD isoenzymes appeared in response to salt stress (Rahnama and Ebrahimzadeh, 2005). In our study, the decrease of isoenzyme activity was observed in SOD in the stems, and CAT in the leaves and roots. In contrast, isoenzyme activity of SOD was activated in the roots and POD was elevated in the leaves and roots. Meanwhile, new isoenzymes were induced in SOD in the roots, and in POD in stems and roots, respectively. Thus, our data indicated that the effect of salt stress on antioxidant enzymes differs depending on the tissues tested. And the induction of stress-related

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Fig. 7. Band pattern of proteins from the leaves (A) and roots (C) of B. papyrifera seedlings resolved by SDS-PAGE. Position of molecular marker proteins (M) was shown on the left side of the gels. (B, D) Densitometric scanning of lanes in A and B. The changed protein bands under NaCl stress were indicated by arrows.

isoenzymes is probably related to the level of ROS, which cause oxidative damage to the plant cells. Studies have revealed that protein synthesis could be affected by salt stress (Hurkman and Tanaka, 1987; Sousa et al., 2003). In our study, changes in protein profiles were observed under NaCl stress which was characterized by the induction of new protein bands as well as diminish of the previously present protein bands. In addition, the increase or decrease of the intensity of protein bands in response to NaCl stress were also found. It was reported that in the seedlings of B. parviflora varying levels of NaCl caused decrease in the intensity of several protein bands, which was roughly proportional to NaCl concentration (Parida et al., 2004b). In pigeonpea [Cajanus cajan (L.) Millsp.], NaCl treatment induced 95.6 kDa protein and 67.5 kDa protein in two genotypes, respectively (Bishnoi et al., 2006). It was considered that changes in protein profiles induced by NaCl may be that the translation of the mRNAs is inhibited or stimulated by increased NaCl concentrations, or it is due to regulation of mRNA transcription (Hurkman and Tanaka, 1987), whether this is the case needs further study. Others suggested that the “disappeared” proteins in response to

salt stress were a result of their denaturation (Bishnoi et al., 2006). Proteins accumulated in salt-stressed plants may provide storage form of nitrogen for the reutilization when stress is over (Singh et al., 1987; Qasim et al., 2003). These proteins may be synthesized de novo in response to salt stress or the increase of presently consecutive expression proteins when plants are exposed to salt stress (Qasim et al., 2003). Another interesting finding in the current work was that obvious differences in ions, antioxidant enzymes and other proteins in response to salt stress were observed between leaves, stems and roots. Higher Na+ accumulation in the roots was detected under high NaCl concentration in comparison with the stems and leaves. It has been suggested that plants can develop salt tolerance or adaptation by reducing the transmission of Na+ from the roots to shoot and leaves (Yasar et al., 2006). Excessive Na+ in the roots can then be sequestered into the vacuoles via proton pumps on the tonoplast to maintain low cytoplasmic Na+ concentrations. In agreement with this assumption, the activity of the plant proton pump, vacuolar H+-ATPase, was found to be significantly stimulated in B. papyrifera by NaCl stress (our unpublished data). The distinction in antioxidant enzyme activities between leaves,

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stems and roots might be due to the difference in structure and functions of these tissues, or might be associated with the marked differences among the types of cellular metabolism existing in these organs (Cavalcanti et al., 2007). Altogether, these results indicated that under salinity conditions the leaves, stems and roots employed distinct mechanisms to cope with salt stress (Qing et al., 2009). Taken together, this study provides information on physiological and biochemical bases of salt stress on woody plants. It may strengthen our understanding of the mechanisms by which salinity affects plant growth and development. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.sajb.2012.11.005.

Acknowledgments This work was supported by National Science & Technology Program in the Twelfth Five-year Plan (No. 2011BAD38B0205), Scientific Research Fund in Jiangsu Province (BK2011872), 2010 Enterprise Doctor Assemblage Program in Jiangsu Province, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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