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Exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant enzyme activities
ARTICLE IN PRESS Journal of Plant Physiology 164 (2007) 553—561
www.elsevier.de/jplph
Exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant enzyme activities Md. Anamul Hoque, Eiji Okuma, Mst. Nasrin Akhter Banu, Yoshimasa Nakamura, Yasuaki Shimoishi, Yoshiyuki Murata Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan Received 17 January 2006; accepted 24 March 2006
KEYWORDS Antioxidant enzymes; Betaine; Proline; Reactive oxygen species; Salt stress
Summary Proline and betaine accumulate in plant cells under environmental stresses including salt stress. Here, we investigated effects of proline and betaine on the growth and activities of antioxidant enzymes in tobacco Bright Yellow-2 (BY-2) culture cells in suspension under salt stress. Both proline and betaine mitigated the inhibition of growth of BY-2 cells under salt stress and the mitigating effect of proline was more than that of betaine. Salt stress significantly decreased the activities of superoxide dismutase (SOD), catalase and peroxidase in BY-2 cells. Exogenous application of proline or betaine alleviated the reduction in catalase and peroxidase activities but not SOD activity under salt stress. In addition, proline was found to be effective in alleviating the inhibition of salt stress-induced catalase and peroxidase activities in BY-2 cells. Neither proline nor betaine directly scavenged superoxide (O 2 ) or hydrogen peroxide (H2O2). It is concluded that exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine because of its superior ability to increase the activities of antioxidant enzymes. & 2006 Elsevier GmbH. All rights reserved.
Introduction Compatible solutes such as proline and betaine are well known to play a major role in the process Corresponding author. Tel.: +81 86 251 8310;
fax: +81 86 251 8388. E-mail address: [email protected] (Y. Murata).
of osmotic adjustment in many different organisms including higher plants (Flowers et al., 1977; Greenway and Munns, 1980; Rhodes and Hanson, 1993; Hasegawa et al., 2000). Most plant species can accumulate proline, while several plant species including important crop plants cannot accumulate betaine because of a deficit in the enzymes involved in betaine biosynthesis (Rathinasabapathi
0176-1617/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2006.03.010
ARTICLE IN PRESS 554 et al., 1993; Holmstro ¨m et al., 2000). The accumulation of proline is essential for plants under osmotic stress (Nanjo et al., 1999), and salt stress up-regulates the key enzyme, P5CS, for proline biosynthesis in Arabidopsis (Hare et al., 1999). Proline and betaine have been considered as osmoprotectants as well as compatible solutes (Le-Rudulier et al., 1984; Csonka and Hanson, 1991). However, the concentrations of proline and betaine are not high enough to adjust the osmotic potential in some plants under stress. Neither glutamic acid, which is a precursor of proline, nor alanine can replace proline in restoring growth of tobacco culture cells, which suggests that proline does not act as nitrogen source and might have another protective role against salt stress (Okuma et al., 2000). It is expected that proline and betaine act as free radical scavengers and/or enzyme protectants as well as compatible solutes. It has been suggested that proline acts as a free radical scavenger and an enzyme protectant (Tsugane et al., 1999; Hong et al., 2000; Okuma et al., 2000, 2002). Okuma et al. (2004) suggests that proline acts as a free radical scavenger to alleviate salt stress, while betaine acts only as a simple osmolyte. It is also reported that proline and betaine protect higher plants against salt/osmotic stresses, not only by adjusting osmotic pressure (Pollard and Wyn Jones, 1979), but also by stabilizing many functional units such as complex II electron transport (Hamilton and Heckathorn, 2001), membranes and proteins (Paleg et al., 1984; Lee et al., 1997; Hare et al., 1998; Mansour, 1998; McNeil et al., 1999), and enzymes such as RUBISCO (Ma ¨kela ¨ et al., 2000). Exogenous proline and betaine mitigate the detrimental effects of Na+ (Harinasut et al., 1996; Okuma et al., 2000). Both proline and betaine mitigated the inhibition of growth of tobacco cells under saline conditions but the mitigating effect of proline was more pronounced than that of betaine (Okuma et al., 2004). Moreover, exogenous application of proline stimulates growth of cells (Kumar and Sharma, 1989) and plants (Fedina et al., 1993; Hamed and Wakeel, 1994), improves metabolism (Alia et al., 1991; Rana and Rana, 1996) and reduces oxidation of the lipid membranes (Jain et al., 2001; Okuma et al., 2004) under stress conditions. Exogenous application of betaine also improves the growth, survival and tolerance of a wide variety of accumulator/non-accumulator plants under various stress conditions (Harinasut et al., 1996; Rajasekaran et al., 1997; Diaz-Zorita et al., 2001). Several investigations have demonstrated that exposure of plants to environmental stresses
Md. Anamul Hoque et al. including salt stress can increase the production of reactive oxygen species (ROS) such as singlet oxygen (O2), superoxide radical (O 2 ), hydrogen peroxide (H2O2) and hydroxyl radical (OH). These ROS are so reactive that they seriously disrupt normal metabolism of plants through oxidation of membrane lipids, proteins and nucleic acids if plants do not have sufficient protective mechanism (Smirnoff, 1993; Go ´mez et al., 1999; Herna ´ndez et al., 2001). Plants possess an antioxidant system that includes antioxidant enzymes such as superoxide dismutase (SOD), catalase and peroxidase to protect their cells against ROS. The protecting roles of proline and betaine in plant cells under salt stress have already been reported (Sairam et al., 2002; Khedr et al., 2003; Demiral and Tu ¨rkan, 2004). Khedr et al. (2003) found that severe salt stress inhibited the activities of antioxidant enzymes catalase and peroxidase but the activities of these enzymes were significantly higher in the presence of proline than in its absence. However, the mechanism for salt tolerance remains elusive. The functions of proline and betaine under stress conditions are yet to be fully understood, though proline but not betaine displays an antioxidant activity (Okuma et al., 2004). Most studies with proline and betaine have focused on their physiological roles and biosynthesis pathways. A few studies have compared the effects of proline and betaine on the activities of antioxidant enzymes in plant cells under salt stress condition. In view of these results, we assessed antioxidant activities of proline and betaine and investigated the activities of antioxidant enzymes in tobacco Bright Yellow-2 (BY-2) suspension cells under salt stress in the presence or absence of proline or betaine to compare protective effects of proline with those of betaine.
Materials and methods Culture of tobacco BY-2 cells Suspension cultured cells of Nicotiana tabacum L., cv. BY-2 were used as the sources of NaClunadapted cell lines (Murata et al., 1994a, b). The standard medium was a modified LS medium (Linsmaier and Skoog, 1965) in which the levels of KH2PO4 and thiamine-HCl were increased to 370 and 1 mg L1, respectively, supplemented with 3% sucrose and 1 mM 2,4-dichlorophenoxyacetic acid (Nagata et al., 1981). The NaCl medium was the standard medium supplemented with 200 mM NaCl. The proline medium was the NaCl medium supplemented with 20 mM proline and the betaine
ARTICLE IN PRESS Proline increases antioxidant enzyme activities more than betaine medium was the NaCl medium supplemented with 20 mM betaine. The cells were cultured and maintained as described by Murata et al. (1994a, b).
Cell growth assay Two grams fresh weights of BY-2 cells, which had been taken from 7-d-old suspension cells, were inoculated into 30 mL of fresh medium, and cultured on a rotary shaker (100 rpm) at 25 1C in the dark. The fresh weight of the cells at 0, 1 and 7 d after inoculation was determined as described previously (Murata et al., 1994a, b). In order to obtain disjointed cells, the cells were incubated in an enzyme solution adjusted to pH 5.5 that contained 1% cellulase Onozuka RS (Yakult Honsha Co. Ltd., Tokyo, Japan), and 0.1% pectolyase Y-23 (Seishin Pharmaceutical Co. Ltd., Tokyo, Japan) at 30 1C for about 30 min. The number of living and round cells was counted with a Haemacytometer to determine the number of cells per flask.
Isolation and preparation of protoplasts To assay the protein content and activities of antioxidant enzymes, protoplasts were enzymatically isolated from cells as described by Murata et al. (1994a, b). The cells were subjected to occasional gentle swirling at 30 1C for about 1 h in an enzyme solution adjusted to pH 5.5 that contained 1% cellulase Onozuka RS, 0.1% pectolyase Y-23 and 0.6 M sorbitol for the cells grown in the standard medium, 1.0 M sorbitol for the cells grown in the NaCl medium and 0.8 M sorbitol for the cells grown in the proline or betaine medium. Protoplasts were collected by centrifugation at 100g for 1 min. The number of protoplasts in the suspension was counted with a Haemacytometer.
Assay of antioxidant enzyme activities Protoplasts were broken up with a sonicator to assay the activity of antioxidant enzymes, SOD (EC: 1.15.1.1), catalase (EC: 1.11.1.6) and peroxidase (EC: 1.11.1.7). The solution containing broken protoplasts hereafter is referred to as the ‘‘sample solution’’. Protein contents were measured as described by Bradford (1976). SOD activity was measured by using an SOD Assay Kit-WST (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). For each SOD activity measurement, 20 mL of the sample solution was placed in the wells for sample and blank 2. Twenty micro-
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liters of distilled water was placed in the wells for blanks 1 and 3. Two hundred microliters of WST working solution was added to each well, and 20 mL of dilution buffer was added to the wells for blanks 2 and 3. Twenty microliters of enzyme working solution was added to the wells for sample and blank 1 and then mixed thoroughly. The absorbance was measured at 450 nm using a microplate reader (model 680, Nippon Bio-Rad Laboratories, Tokyo, Japan) after incubation at 37 1C for 20 min. The activity was calculated (inhibition rate %) using the following equation: SOD activity (inhibition rate %) ¼ {[(A blank 1A blank 3)–(A sampleA blank 2)]/(A blank 1A blank 3)} 100. SOD activity was expressed as a percentage, where the activity of non-stressed cells was 100%. Catalase activity was measured according to Johansson and Borg (1988). The reaction buffer solution consisted of 250 mM KH2PO4–NaOH buffer (pH 7.0), 5.9 M methanol and 4.2 mM H2O2. The reaction was initiated by the addition of 300 mL of sample solution to the reaction buffer solution. The reaction mixture was incubated for 20 min at room temperature. The reaction was terminated by the addition of 150 mL of 7.8 M KOH. Thereafter, 300 mL of 34.2 mM purpald in 480 mM HCl was added and a second incubation was performed for 10 min at room temperature. The product of the reaction between formaldehyde and purpald was oxidized by adding 150 mL of 65.2 mM potassium periodate in 470 mM KOH. The solution was centrifuged at 9500g for 10 min and the absorbance of the supernatant was measured at 550 nm. Standard solutions of formaldehyde were used for the calculation of enzyme activity and the activity was expressed as units mg1 protein. One unit of activity was defined as nmol H2O2 decomposed per min. Peroxidase activity was determined according to Nakano and Asada (1981). The reaction buffer solution contained 50 mM KH2PO4 buffer (pH 7.0), 0.1 mM EDTA, 0.1 mM H2O2 and 10 mM guaiacol. The reaction was started by the addition of the sample solution to the reaction buffer solution. The activity was calculated from change in absorbance at 470 nm for 30 s where an extinction coefficient is 26.6 mM1 cm1 and was expressed as units mg1 protein. One unit of activity was defined as the formation of mmol tetraguaiacol per min.
Determination of O 2 degradation An SOD assay kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used for measuring the ability to degrade O 2 by proline or betaine. The absorbance of the reaction solution was
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Determination of H2O2 degradation The degradation of H2O2 by proline and betaine was investigated as follows. The assay solution contained 0.015% H2O2 and 300 mM proline or betaine. Each solution was kept at room temperature for 12 h. The solution was then titrated with acidic 0.1 N KMnO4 with sulfuric acid to determine the remaining H2O2.
Statistical analysis The significance of differences between mean values was compared by Student’s T-test. Differences at Po0:05 were considered significant.
Results Growth characteristics Figure 1 shows fresh weights of tobacco BY-2 cells cultured in the standard, NaCl, proline and betaine media. The fresh weights of cells in the NaCl medium were significantly lower than those in the standard medium at 0, 1 and 7 d after inoculation. The fresh weights of cells in the proline or betaine medium were the same as those of cells in the NaCl medium at 0 and 1 d after inoculation, showing that 12 a
Fresh weight (g flask-1)
10
0 d after inoculation 1 d after inoculation 7 d after inoculation
8 6 4
b c
c c
2 0
d d d
Non - stress
NaCl
d d
NaCl + proline
d d
NaCl + betaine
Figure 1. Fresh weight of NaCl-unadapted tobacco BY-2 suspension cells at 0, 1 and 7 d after inoculation. Nonstress, NaCl, NaCl+proline and NaCl+betaine indicate the standard medium, the NaCl medium, the proline medium and the betaine medium, respectively. Values represent the mean7SD (n ¼ 3). Bars with the same letters are not significantly different at Po0:05.
4.5 a
4 Number of cells x 107 flask-1
measured at 450 nm using a microplate reader (model 680, Nippon Bio-Rad Laboratories, Tokyo, Japan).
0 d after inoculation 1 d after inoculation 7 d after inoculation
3.5 3 2.5
b
2 1.5
c
bc
bc d d d
1
d
d
d d
0.5 0 Non-stress
NaCl
NaCl + proline
NaCl + betaine
Figure 2. Number of living cells per flask in NaClunadapted tobacco BY-2 suspension culture at 0, 1 and 7 d after inoculation. Non-stress, NaCl, NaCl+proline and NaCl+betaine indicate the standard medium, the NaCl medium, the proline medium and the betaine medium, respectively. Values represent the mean7SD (n ¼ 3). Bars with the same letters are not significantly different at Po0:05.
neither proline nor betaine mitigated the reduction in fresh weight of BY-2 cells under salt stress at 0 and 1 d after inoculation. However, the fresh weight of cells in the proline or betaine medium was significantly higher than that in the NaCl medium at 7 d after inoculation. In addition, the fresh weight of cells was significantly higher in the proline medium than in the betaine medium. Figure 2 shows the number of tobacco BY-2 cells cultured in the standard, NaCl, proline and betaine media. The numbers of cells in the NaCl medium were significantly lower than those in the standard medium. The numbers of cells did not change in the NaCl medium at 0 and 1 d after inoculation even though 20 mM proline or betaine was added to the NaCl medium. However, the numbers of cells in the proline and betaine media increased and were significantly greater than those in the NaCl medium at 7 d after inoculation. The number of cells was greater in the proline medium than in the betaine medium but the difference was not significant.
Antioxidant enzyme activities SOD activities of tobacco BY-2 cells cultured in the standard, NaCl, proline and betaine media are shown in Fig. 3. It shows that SOD activity of BY-2 cells was significantly decreased under salt stress. Exogenous application of proline increased SOD activity in BY-2 cells under NaCl stress but this increase was not significant, whereas exogenous
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Relative SOD activity (%)
a
100 80 60
b
40
b
b
20 0 Non-stress
NaCl
NaCl + proline
NaCl + betaine
Figure 3. Superoxide dismutase (SOD) activity of NaClunadapted tobacco BY-2 suspension cells induced by proline and betaine under NaCl stress. Non-stress, NaCl, NaCl+proline and NaCl+betaine indicate the standard medium, the NaCl medium, the proline medium and the betaine medium, respectively. Values represent the mean7SD (n ¼ 529). Bars with the same letters are not significantly different at Po0:05.
Catalase activity (units mg-1 protein)
6 5 a
4 3
a a
2 1
b
0 Non-stress
NaCl
NaCl + proline
NaCl + betaine
Figure 4. Catalase activity of NaCl-unadapted tobacco BY-2 suspension cells induced by proline and betaine under NaCl stress. Non-stress, NaCl, NaCl+proline and NaCl+betaine indicate the standard medium, the NaCl medium, the proline medium and the betaine medium, respectively. Values represent the mean7SD (n ¼ 5). Bars with the same letters are not significantly different at Po0:05.
application of betaine did not affect SOD activity in BY-2 cells under NaCl stress. NaCl stress significantly decreased the catalase activity in tobacco BY-2 cells as represented in Fig. 4. The activity in non-stressed cells was 3.5fold higher than that in NaCl-stressed cells. Exogenous proline and betaine showed a significant increase in catalase activity of NaCl-stressed cells.
Peroxidase activity (units mg-1 protein)
Proline increases antioxidant enzyme activities more than betaine
557
0.6 0.5 a
a
0.4 0.3 0.2 b
0.1
b
0 Non-stress
NaCl
NaCl + proline
NaCl + betaine
Figure 5. Peroxidase activity of NaCl-unadapted tobacco BY-2 suspension cells induced by proline and betaine under NaCl stress. Non-stress, NaCl, NaCl+proline and NaCl+betaine indicate the standard medium, the NaCl medium, the proline medium and the betaine medium, respectively. Values represent the mean7SD (n ¼ 426). Bars with the same letters are not significantly different at Po0:05.
In comparison with the cells cultured in the NaCl medium, the cells cultured in the proline medium showed 6-fold higher catalase activity and the cells cultured in the betaine medium showed 2.3-fold higher activities. The effect of proline on catalase activity of NaCl-stressed cells was more pronounced than that of betaine. The catalase activity in the proline medium was higher than that in the standard medium. NaCl stress also significantly decreased the peroxidase activity in BY-2 cells as shown in Fig. 5. Non-stressed cells had approximately 6-fold higher peroxidase activity than NaCl-stressed cells. Addition of proline and betaine to the NaCl medium increased the peroxidase activity in BY-2 cells. Proline caused a significant increase in peroxidase activity, whereas betaine did not show significant effect on peroxidase activity. In NaCl-stressed cells, peroxidase activity was 6.3-fold higher in the presence of proline than in its absence. There was no difference in peroxidase activity between cells grown in the standard medium and in the proline medium, whereas the activity in the standard medium was significantly higher than that in the betaine medium.
Radical scavenging activity of proline and betaine We investigated whether proline or betaine scavenged superoxide (O 2 ) radical and broke down
ARTICLE IN PRESS 558 hydrogen peroxide (H2O2) in the absence of any kind of enzyme. It was found that neither proline nor betaine significantly degraded O 2 and H2O2 under the conditions used here (data not shown).
Discussion Higher plants accumulate proline and betaine under salt stress. Okuma et al. (2004) reported that both proline and betaine mitigated the inhibition of growth of tobacco BY-2 cells under saline conditions, that proline displayed an antioxidant activity and improved the growth of cells under saline conditions more than betaine did. The fresh weight decreased to about 1 g at 0 and 1 d after inoculation of 2 g BY-2 cells into the NaCl medium irrespective of the presence or absence of proline and betaine, suggesting salt-induced hyperosmotic stress causes loss of water from cells (i.e., cell shrinkage). Exogenous proline and betaine significantly increased the fresh weight (Fig. 1) and the number (Fig. 2) of BY-2 cells under salt stress at 7 d after inoculation, and proline was found to be more effective than betaine in mitigating the inhibition of growth of BY-2 cells under salt stress. Our results are consistent with the results of Jain et al. (2001) who found that addition of proline to the culture medium reduced the salt stress-induced decline of fresh weight in cell lines of groundnut. Exogenous proline reduces the amount of malondialdehyde in salt-stressed cells but exogenous betaine does not reduce the malondialdehyde amount (Okuma et al., 2004). These results suggest that the difference in the mitigation effects between proline and betaine may be responsible for the difference in the antioxidant activity. Plant tissues contain several antioxidant enzymes such as SOD, catalase and peroxidase to scavenge ROS. It has been reported that antioxidant enzyme activities decrease in plant cells under salt stress (Shalata et al., 2001; Khedr et al., 2003; Mishra and Das, 2003; Mittova et al., 2004) and that antioxidant enzyme activities increase in the presence of proline (Khedr et al., 2003; Chen and Dickman, 2005). An increase in the activity of antioxidant enzymes alleviates salt stress. SOD activity directly modulates the amount of ROS and higher SOD activity contributes to detoxification of superoxide under salt stress. Results in this study showed that SOD activity in NaCl-stressed cells was significantly lower than that in nonstressed cells, whereas Go ´mez et al. (2004) found increases in all SOD isoenzymes of pea chloroplasts in long-term NaCl treatment. Some researchers
Md. Anamul Hoque et al. also suggest that salt stress leads to a decrease in SOD activity in salt-sensitive plants but to an increase in salt-tolerant ones (Shalata and Tal, 1998; Sreenivasulu et al., 2000; Rout and Shaw, 2001). For example, salt stress was found to increase SOD activity in a salt-tolerant cultivar of tomato (Harinasut et al., 2003; Mittova et al., 2003) and to decrease it in salt-sensitive varieties of rice (Dionisio-Sese and Tobita, 1998). Results in this study revealed that salt stress did not increase SOD activity in NaCl-unadapted tobacco BY-2 cells, even with exogenous application of proline or betaine. On the other hand, Chen and Dickman (2005) suggest that addition of proline does not increase SOD activity during oxidative stress. These results have good agreement with the results of Demiral and Tu ¨rkan (2004) who found that salt treatment increased SOD activity in both salt-tolerant and saltsensitive rice cultivars but glycinebetaine did not change significantly SOD activity in salt-sensitive rice cultivars under salt stress. Taken together, these results suggest that neither proline nor betaine could enhance SOD activity to scavenge superoxide under salt stress. Salinity accumulates the ROS including H2O2 in plant cells. The metabolism of H2O2 is dependent on various functionally interrelated antioxidant enzymes such as catalases and peroxidases localized in almost all compartments of plant cells. The increase in antioxidant enzyme activities is involved in eliminating H2O2 from salt-stressed roots (Kim et al., 2005). Our results demonstrated that catalase and peroxidase activities in NaCl-unadapted BY-2 cells decreased when the cells were cultured in the NaCl medium. These results are also supported by the findings of Shalata et al. (2001) and Mittova et al. (2004). The reductions in catalase and peroxidase activities suggest that these enzymes were unable to completely detoxify H2O2 generated by salt stress. Under salt stress conditions, catalase (Fig. 4) and peroxidase (Fig. 5) activities increased significantly in the presence of proline. These results are consistent with the results of Khedr et al. (2003) who observed that the activities of catalase and peroxidase decreased under severe salt stress but increased in the presence of proline. Demiral and Tu ¨rkan (2004) found that salt stress decreased the activities of catalase and peroxidase in both saltsensitive and salt-tolerant rice varieties, while salt stress increased the activities of these enzymes in the presence of betaine. However, results obtained in this study show that exogenous betaine increased significantly the activities of catalase but did not increase significantly the activities of peroxidase in BY-2 cells under salt stress. Catalase and
ARTICLE IN PRESS Proline increases antioxidant enzyme activities more than betaine peroxidase play an important role in the fine regulation of ROS concentrations in cells (Elstner, 1987). Proline and betaine have been considered to act as compatible solutes, osmoprotectants and hydroxyl radical scavengers (see Introduction). The present investigation suggests that both exogenous proline and betaine are able to effectively detoxify H2O2 by enhancing the activities of catalase and peroxidase under salt stress. Exogenous proline is more capable than exogenous betaine in enhancing the activities of catalase and peroxidase under salt stress. In this study, a positive relationship among proline, betaine and up-regulation of antioxidant enzyme activities in tobacco BY-2 cells has been observed under salt stress. Therefore, we conclude that up-regulation of antioxidant enzyme activities by proline and betaine scavenges ROS and thereby mitigates the detrimental effects of salt stress. Proline is more effective than betaine in mitigating the detrimental effects of salt stress by upregulation of antioxidant enzyme activities.
Acknowledgments The authors are grateful to Japan Tobacco Inc. for the generous gift of Nicotiana tabacum L. cv. Bright Yellow cultured cells in suspension.
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Md. Anamul Hoque et al. suspension after adaptation to 50 mM NaCl. Plant Cell Physiol 1994a;35:87–92. Murata Y, Obi I, Yoshihashi M, Ikeda T, Kakutani T. Salt adaptation of K+ channels in the plasma membrane of tobacco cells in suspension culture. Plant Cell Physiol 1994b;35:637–44. Nagata T, Okada K, Takebe I, Matsui C. Delivery of tobacco mosaic virus RNA into plant protoplasts mediated by reverse-phase evaporation vesicles (liposomes). Mol Gen Genet 1981;184:161–5. Nakano Y, Asada K. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 1981;22:867–80. Nanjo T, Kobayashi TM, Yoshida Y, Kakubari Y, YamaguchiShinozaki K, Shinozaki K. Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana. FEBS Lett 1999;461:205–10. Okuma E, Soeda K, Tada M, Murata Y. Exogenous proline mitigates the inhibition of growth of Nicotiana tabacum cultured cells under saline conditions. Soil Sci Plant Nutr 2000;46:257–63. Okuma E, Soeda K, Fukuda M, Tada M, Murata Y. Negative correlation between the ratio of K+ to Na+ and proline accumulation in tobacco suspension cells. Soil Sci Plant Nutr 2002;48:753–7. Okuma E, Murakami Y, Shimoishi Y, Tada M, Murata Y. Effects of exogenous application of proline and betaine on the growth of tobacco cultured cells under saline conditions. Soil Sci Plant Nutr 2004;50:1301–5. Paleg LG, Stewart GR, Bradbeer JW. Proline and glycine betaine influence protein solvation. Plant Physiol 1984;75:974–8. Pollard A, Wyn Jones RG. Enzyme activities in concentrated solutions of glycinebetaine and other solutes. Planta 1979;144:291–8. Rajasekaran LR, Kriedemann PE, Aspinall D, Paleg LG. Physiological significance of proline and glycinebetaine: maintaining photosynthesis during NaCl stress in wheat. Photosynthetica 1997;34:357–66. Rana VK, Rana U. Modulation of calcium uptake by exogenous amino acids in Phaseolus vulgaris seedlings. Acta Physiol Plant 1996;18:117–20. Rathinasabapathi B, Gage DA, Mackill DJ, Hanson AD. Cultivated and wild rices do not accumulate glycinebetaine due to deficiencies in two biosynthetic steps. Crop Sci 1993;33:534–8. Rhodes D, Hanson AD. Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu Rev Plant Physiol Plant Mol Biol 1993;44:357–84. Rout NP, Shaw BP. Salt tolerance in aquatic macrophytes: possible involvement of the antioxidative enzymes. Plant Sci 2001;160:415–23. Sairam RK, Rao KV, Srivastava GC. Differential response of wheat genotypes to long-term salinity stress in relation to oxidative stress, antioxidant activity and osmolytes concentration. Plant Sci 2002;163: 1037–46. Shalata A, Tal M. The effect of salt stress on lipid peroxidation and antioxidants in the leaf of the
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Exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant enzyme activities Md. Anamul Hoque, Eiji Okuma, Mst. Nasrin Akhter Banu, Yoshimasa Nakamura, Yasuaki Shimoishi, Yoshiyuki Murata Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan Received 17 January 2006; accepted 24 March 2006
KEYWORDS Antioxidant enzymes; Betaine; Proline; Reactive oxygen species; Salt stress
Summary Proline and betaine accumulate in plant cells under environmental stresses including salt stress. Here, we investigated effects of proline and betaine on the growth and activities of antioxidant enzymes in tobacco Bright Yellow-2 (BY-2) culture cells in suspension under salt stress. Both proline and betaine mitigated the inhibition of growth of BY-2 cells under salt stress and the mitigating effect of proline was more than that of betaine. Salt stress significantly decreased the activities of superoxide dismutase (SOD), catalase and peroxidase in BY-2 cells. Exogenous application of proline or betaine alleviated the reduction in catalase and peroxidase activities but not SOD activity under salt stress. In addition, proline was found to be effective in alleviating the inhibition of salt stress-induced catalase and peroxidase activities in BY-2 cells. Neither proline nor betaine directly scavenged superoxide (O 2 ) or hydrogen peroxide (H2O2). It is concluded that exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine because of its superior ability to increase the activities of antioxidant enzymes. & 2006 Elsevier GmbH. All rights reserved.
Introduction Compatible solutes such as proline and betaine are well known to play a major role in the process Corresponding author. Tel.: +81 86 251 8310;
fax: +81 86 251 8388. E-mail address: [email protected] (Y. Murata).
of osmotic adjustment in many different organisms including higher plants (Flowers et al., 1977; Greenway and Munns, 1980; Rhodes and Hanson, 1993; Hasegawa et al., 2000). Most plant species can accumulate proline, while several plant species including important crop plants cannot accumulate betaine because of a deficit in the enzymes involved in betaine biosynthesis (Rathinasabapathi
0176-1617/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2006.03.010
ARTICLE IN PRESS 554 et al., 1993; Holmstro ¨m et al., 2000). The accumulation of proline is essential for plants under osmotic stress (Nanjo et al., 1999), and salt stress up-regulates the key enzyme, P5CS, for proline biosynthesis in Arabidopsis (Hare et al., 1999). Proline and betaine have been considered as osmoprotectants as well as compatible solutes (Le-Rudulier et al., 1984; Csonka and Hanson, 1991). However, the concentrations of proline and betaine are not high enough to adjust the osmotic potential in some plants under stress. Neither glutamic acid, which is a precursor of proline, nor alanine can replace proline in restoring growth of tobacco culture cells, which suggests that proline does not act as nitrogen source and might have another protective role against salt stress (Okuma et al., 2000). It is expected that proline and betaine act as free radical scavengers and/or enzyme protectants as well as compatible solutes. It has been suggested that proline acts as a free radical scavenger and an enzyme protectant (Tsugane et al., 1999; Hong et al., 2000; Okuma et al., 2000, 2002). Okuma et al. (2004) suggests that proline acts as a free radical scavenger to alleviate salt stress, while betaine acts only as a simple osmolyte. It is also reported that proline and betaine protect higher plants against salt/osmotic stresses, not only by adjusting osmotic pressure (Pollard and Wyn Jones, 1979), but also by stabilizing many functional units such as complex II electron transport (Hamilton and Heckathorn, 2001), membranes and proteins (Paleg et al., 1984; Lee et al., 1997; Hare et al., 1998; Mansour, 1998; McNeil et al., 1999), and enzymes such as RUBISCO (Ma ¨kela ¨ et al., 2000). Exogenous proline and betaine mitigate the detrimental effects of Na+ (Harinasut et al., 1996; Okuma et al., 2000). Both proline and betaine mitigated the inhibition of growth of tobacco cells under saline conditions but the mitigating effect of proline was more pronounced than that of betaine (Okuma et al., 2004). Moreover, exogenous application of proline stimulates growth of cells (Kumar and Sharma, 1989) and plants (Fedina et al., 1993; Hamed and Wakeel, 1994), improves metabolism (Alia et al., 1991; Rana and Rana, 1996) and reduces oxidation of the lipid membranes (Jain et al., 2001; Okuma et al., 2004) under stress conditions. Exogenous application of betaine also improves the growth, survival and tolerance of a wide variety of accumulator/non-accumulator plants under various stress conditions (Harinasut et al., 1996; Rajasekaran et al., 1997; Diaz-Zorita et al., 2001). Several investigations have demonstrated that exposure of plants to environmental stresses
Md. Anamul Hoque et al. including salt stress can increase the production of reactive oxygen species (ROS) such as singlet oxygen (O2), superoxide radical (O 2 ), hydrogen peroxide (H2O2) and hydroxyl radical (OH). These ROS are so reactive that they seriously disrupt normal metabolism of plants through oxidation of membrane lipids, proteins and nucleic acids if plants do not have sufficient protective mechanism (Smirnoff, 1993; Go ´mez et al., 1999; Herna ´ndez et al., 2001). Plants possess an antioxidant system that includes antioxidant enzymes such as superoxide dismutase (SOD), catalase and peroxidase to protect their cells against ROS. The protecting roles of proline and betaine in plant cells under salt stress have already been reported (Sairam et al., 2002; Khedr et al., 2003; Demiral and Tu ¨rkan, 2004). Khedr et al. (2003) found that severe salt stress inhibited the activities of antioxidant enzymes catalase and peroxidase but the activities of these enzymes were significantly higher in the presence of proline than in its absence. However, the mechanism for salt tolerance remains elusive. The functions of proline and betaine under stress conditions are yet to be fully understood, though proline but not betaine displays an antioxidant activity (Okuma et al., 2004). Most studies with proline and betaine have focused on their physiological roles and biosynthesis pathways. A few studies have compared the effects of proline and betaine on the activities of antioxidant enzymes in plant cells under salt stress condition. In view of these results, we assessed antioxidant activities of proline and betaine and investigated the activities of antioxidant enzymes in tobacco Bright Yellow-2 (BY-2) suspension cells under salt stress in the presence or absence of proline or betaine to compare protective effects of proline with those of betaine.
Materials and methods Culture of tobacco BY-2 cells Suspension cultured cells of Nicotiana tabacum L., cv. BY-2 were used as the sources of NaClunadapted cell lines (Murata et al., 1994a, b). The standard medium was a modified LS medium (Linsmaier and Skoog, 1965) in which the levels of KH2PO4 and thiamine-HCl were increased to 370 and 1 mg L1, respectively, supplemented with 3% sucrose and 1 mM 2,4-dichlorophenoxyacetic acid (Nagata et al., 1981). The NaCl medium was the standard medium supplemented with 200 mM NaCl. The proline medium was the NaCl medium supplemented with 20 mM proline and the betaine
ARTICLE IN PRESS Proline increases antioxidant enzyme activities more than betaine medium was the NaCl medium supplemented with 20 mM betaine. The cells were cultured and maintained as described by Murata et al. (1994a, b).
Cell growth assay Two grams fresh weights of BY-2 cells, which had been taken from 7-d-old suspension cells, were inoculated into 30 mL of fresh medium, and cultured on a rotary shaker (100 rpm) at 25 1C in the dark. The fresh weight of the cells at 0, 1 and 7 d after inoculation was determined as described previously (Murata et al., 1994a, b). In order to obtain disjointed cells, the cells were incubated in an enzyme solution adjusted to pH 5.5 that contained 1% cellulase Onozuka RS (Yakult Honsha Co. Ltd., Tokyo, Japan), and 0.1% pectolyase Y-23 (Seishin Pharmaceutical Co. Ltd., Tokyo, Japan) at 30 1C for about 30 min. The number of living and round cells was counted with a Haemacytometer to determine the number of cells per flask.
Isolation and preparation of protoplasts To assay the protein content and activities of antioxidant enzymes, protoplasts were enzymatically isolated from cells as described by Murata et al. (1994a, b). The cells were subjected to occasional gentle swirling at 30 1C for about 1 h in an enzyme solution adjusted to pH 5.5 that contained 1% cellulase Onozuka RS, 0.1% pectolyase Y-23 and 0.6 M sorbitol for the cells grown in the standard medium, 1.0 M sorbitol for the cells grown in the NaCl medium and 0.8 M sorbitol for the cells grown in the proline or betaine medium. Protoplasts were collected by centrifugation at 100g for 1 min. The number of protoplasts in the suspension was counted with a Haemacytometer.
Assay of antioxidant enzyme activities Protoplasts were broken up with a sonicator to assay the activity of antioxidant enzymes, SOD (EC: 1.15.1.1), catalase (EC: 1.11.1.6) and peroxidase (EC: 1.11.1.7). The solution containing broken protoplasts hereafter is referred to as the ‘‘sample solution’’. Protein contents were measured as described by Bradford (1976). SOD activity was measured by using an SOD Assay Kit-WST (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). For each SOD activity measurement, 20 mL of the sample solution was placed in the wells for sample and blank 2. Twenty micro-
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liters of distilled water was placed in the wells for blanks 1 and 3. Two hundred microliters of WST working solution was added to each well, and 20 mL of dilution buffer was added to the wells for blanks 2 and 3. Twenty microliters of enzyme working solution was added to the wells for sample and blank 1 and then mixed thoroughly. The absorbance was measured at 450 nm using a microplate reader (model 680, Nippon Bio-Rad Laboratories, Tokyo, Japan) after incubation at 37 1C for 20 min. The activity was calculated (inhibition rate %) using the following equation: SOD activity (inhibition rate %) ¼ {[(A blank 1A blank 3)–(A sampleA blank 2)]/(A blank 1A blank 3)} 100. SOD activity was expressed as a percentage, where the activity of non-stressed cells was 100%. Catalase activity was measured according to Johansson and Borg (1988). The reaction buffer solution consisted of 250 mM KH2PO4–NaOH buffer (pH 7.0), 5.9 M methanol and 4.2 mM H2O2. The reaction was initiated by the addition of 300 mL of sample solution to the reaction buffer solution. The reaction mixture was incubated for 20 min at room temperature. The reaction was terminated by the addition of 150 mL of 7.8 M KOH. Thereafter, 300 mL of 34.2 mM purpald in 480 mM HCl was added and a second incubation was performed for 10 min at room temperature. The product of the reaction between formaldehyde and purpald was oxidized by adding 150 mL of 65.2 mM potassium periodate in 470 mM KOH. The solution was centrifuged at 9500g for 10 min and the absorbance of the supernatant was measured at 550 nm. Standard solutions of formaldehyde were used for the calculation of enzyme activity and the activity was expressed as units mg1 protein. One unit of activity was defined as nmol H2O2 decomposed per min. Peroxidase activity was determined according to Nakano and Asada (1981). The reaction buffer solution contained 50 mM KH2PO4 buffer (pH 7.0), 0.1 mM EDTA, 0.1 mM H2O2 and 10 mM guaiacol. The reaction was started by the addition of the sample solution to the reaction buffer solution. The activity was calculated from change in absorbance at 470 nm for 30 s where an extinction coefficient is 26.6 mM1 cm1 and was expressed as units mg1 protein. One unit of activity was defined as the formation of mmol tetraguaiacol per min.
Determination of O 2 degradation An SOD assay kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used for measuring the ability to degrade O 2 by proline or betaine. The absorbance of the reaction solution was
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Determination of H2O2 degradation The degradation of H2O2 by proline and betaine was investigated as follows. The assay solution contained 0.015% H2O2 and 300 mM proline or betaine. Each solution was kept at room temperature for 12 h. The solution was then titrated with acidic 0.1 N KMnO4 with sulfuric acid to determine the remaining H2O2.
Statistical analysis The significance of differences between mean values was compared by Student’s T-test. Differences at Po0:05 were considered significant.
Results Growth characteristics Figure 1 shows fresh weights of tobacco BY-2 cells cultured in the standard, NaCl, proline and betaine media. The fresh weights of cells in the NaCl medium were significantly lower than those in the standard medium at 0, 1 and 7 d after inoculation. The fresh weights of cells in the proline or betaine medium were the same as those of cells in the NaCl medium at 0 and 1 d after inoculation, showing that 12 a
Fresh weight (g flask-1)
10
0 d after inoculation 1 d after inoculation 7 d after inoculation
8 6 4
b c
c c
2 0
d d d
Non - stress
NaCl
d d
NaCl + proline
d d
NaCl + betaine
Figure 1. Fresh weight of NaCl-unadapted tobacco BY-2 suspension cells at 0, 1 and 7 d after inoculation. Nonstress, NaCl, NaCl+proline and NaCl+betaine indicate the standard medium, the NaCl medium, the proline medium and the betaine medium, respectively. Values represent the mean7SD (n ¼ 3). Bars with the same letters are not significantly different at Po0:05.
4.5 a
4 Number of cells x 107 flask-1
measured at 450 nm using a microplate reader (model 680, Nippon Bio-Rad Laboratories, Tokyo, Japan).
0 d after inoculation 1 d after inoculation 7 d after inoculation
3.5 3 2.5
b
2 1.5
c
bc
bc d d d
1
d
d
d d
0.5 0 Non-stress
NaCl
NaCl + proline
NaCl + betaine
Figure 2. Number of living cells per flask in NaClunadapted tobacco BY-2 suspension culture at 0, 1 and 7 d after inoculation. Non-stress, NaCl, NaCl+proline and NaCl+betaine indicate the standard medium, the NaCl medium, the proline medium and the betaine medium, respectively. Values represent the mean7SD (n ¼ 3). Bars with the same letters are not significantly different at Po0:05.
neither proline nor betaine mitigated the reduction in fresh weight of BY-2 cells under salt stress at 0 and 1 d after inoculation. However, the fresh weight of cells in the proline or betaine medium was significantly higher than that in the NaCl medium at 7 d after inoculation. In addition, the fresh weight of cells was significantly higher in the proline medium than in the betaine medium. Figure 2 shows the number of tobacco BY-2 cells cultured in the standard, NaCl, proline and betaine media. The numbers of cells in the NaCl medium were significantly lower than those in the standard medium. The numbers of cells did not change in the NaCl medium at 0 and 1 d after inoculation even though 20 mM proline or betaine was added to the NaCl medium. However, the numbers of cells in the proline and betaine media increased and were significantly greater than those in the NaCl medium at 7 d after inoculation. The number of cells was greater in the proline medium than in the betaine medium but the difference was not significant.
Antioxidant enzyme activities SOD activities of tobacco BY-2 cells cultured in the standard, NaCl, proline and betaine media are shown in Fig. 3. It shows that SOD activity of BY-2 cells was significantly decreased under salt stress. Exogenous application of proline increased SOD activity in BY-2 cells under NaCl stress but this increase was not significant, whereas exogenous
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Relative SOD activity (%)
a
100 80 60
b
40
b
b
20 0 Non-stress
NaCl
NaCl + proline
NaCl + betaine
Figure 3. Superoxide dismutase (SOD) activity of NaClunadapted tobacco BY-2 suspension cells induced by proline and betaine under NaCl stress. Non-stress, NaCl, NaCl+proline and NaCl+betaine indicate the standard medium, the NaCl medium, the proline medium and the betaine medium, respectively. Values represent the mean7SD (n ¼ 529). Bars with the same letters are not significantly different at Po0:05.
Catalase activity (units mg-1 protein)
6 5 a
4 3
a a
2 1
b
0 Non-stress
NaCl
NaCl + proline
NaCl + betaine
Figure 4. Catalase activity of NaCl-unadapted tobacco BY-2 suspension cells induced by proline and betaine under NaCl stress. Non-stress, NaCl, NaCl+proline and NaCl+betaine indicate the standard medium, the NaCl medium, the proline medium and the betaine medium, respectively. Values represent the mean7SD (n ¼ 5). Bars with the same letters are not significantly different at Po0:05.
application of betaine did not affect SOD activity in BY-2 cells under NaCl stress. NaCl stress significantly decreased the catalase activity in tobacco BY-2 cells as represented in Fig. 4. The activity in non-stressed cells was 3.5fold higher than that in NaCl-stressed cells. Exogenous proline and betaine showed a significant increase in catalase activity of NaCl-stressed cells.
Peroxidase activity (units mg-1 protein)
Proline increases antioxidant enzyme activities more than betaine
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0.6 0.5 a
a
0.4 0.3 0.2 b
0.1
b
0 Non-stress
NaCl
NaCl + proline
NaCl + betaine
Figure 5. Peroxidase activity of NaCl-unadapted tobacco BY-2 suspension cells induced by proline and betaine under NaCl stress. Non-stress, NaCl, NaCl+proline and NaCl+betaine indicate the standard medium, the NaCl medium, the proline medium and the betaine medium, respectively. Values represent the mean7SD (n ¼ 426). Bars with the same letters are not significantly different at Po0:05.
In comparison with the cells cultured in the NaCl medium, the cells cultured in the proline medium showed 6-fold higher catalase activity and the cells cultured in the betaine medium showed 2.3-fold higher activities. The effect of proline on catalase activity of NaCl-stressed cells was more pronounced than that of betaine. The catalase activity in the proline medium was higher than that in the standard medium. NaCl stress also significantly decreased the peroxidase activity in BY-2 cells as shown in Fig. 5. Non-stressed cells had approximately 6-fold higher peroxidase activity than NaCl-stressed cells. Addition of proline and betaine to the NaCl medium increased the peroxidase activity in BY-2 cells. Proline caused a significant increase in peroxidase activity, whereas betaine did not show significant effect on peroxidase activity. In NaCl-stressed cells, peroxidase activity was 6.3-fold higher in the presence of proline than in its absence. There was no difference in peroxidase activity between cells grown in the standard medium and in the proline medium, whereas the activity in the standard medium was significantly higher than that in the betaine medium.
Radical scavenging activity of proline and betaine We investigated whether proline or betaine scavenged superoxide (O 2 ) radical and broke down
ARTICLE IN PRESS 558 hydrogen peroxide (H2O2) in the absence of any kind of enzyme. It was found that neither proline nor betaine significantly degraded O 2 and H2O2 under the conditions used here (data not shown).
Discussion Higher plants accumulate proline and betaine under salt stress. Okuma et al. (2004) reported that both proline and betaine mitigated the inhibition of growth of tobacco BY-2 cells under saline conditions, that proline displayed an antioxidant activity and improved the growth of cells under saline conditions more than betaine did. The fresh weight decreased to about 1 g at 0 and 1 d after inoculation of 2 g BY-2 cells into the NaCl medium irrespective of the presence or absence of proline and betaine, suggesting salt-induced hyperosmotic stress causes loss of water from cells (i.e., cell shrinkage). Exogenous proline and betaine significantly increased the fresh weight (Fig. 1) and the number (Fig. 2) of BY-2 cells under salt stress at 7 d after inoculation, and proline was found to be more effective than betaine in mitigating the inhibition of growth of BY-2 cells under salt stress. Our results are consistent with the results of Jain et al. (2001) who found that addition of proline to the culture medium reduced the salt stress-induced decline of fresh weight in cell lines of groundnut. Exogenous proline reduces the amount of malondialdehyde in salt-stressed cells but exogenous betaine does not reduce the malondialdehyde amount (Okuma et al., 2004). These results suggest that the difference in the mitigation effects between proline and betaine may be responsible for the difference in the antioxidant activity. Plant tissues contain several antioxidant enzymes such as SOD, catalase and peroxidase to scavenge ROS. It has been reported that antioxidant enzyme activities decrease in plant cells under salt stress (Shalata et al., 2001; Khedr et al., 2003; Mishra and Das, 2003; Mittova et al., 2004) and that antioxidant enzyme activities increase in the presence of proline (Khedr et al., 2003; Chen and Dickman, 2005). An increase in the activity of antioxidant enzymes alleviates salt stress. SOD activity directly modulates the amount of ROS and higher SOD activity contributes to detoxification of superoxide under salt stress. Results in this study showed that SOD activity in NaCl-stressed cells was significantly lower than that in nonstressed cells, whereas Go ´mez et al. (2004) found increases in all SOD isoenzymes of pea chloroplasts in long-term NaCl treatment. Some researchers
Md. Anamul Hoque et al. also suggest that salt stress leads to a decrease in SOD activity in salt-sensitive plants but to an increase in salt-tolerant ones (Shalata and Tal, 1998; Sreenivasulu et al., 2000; Rout and Shaw, 2001). For example, salt stress was found to increase SOD activity in a salt-tolerant cultivar of tomato (Harinasut et al., 2003; Mittova et al., 2003) and to decrease it in salt-sensitive varieties of rice (Dionisio-Sese and Tobita, 1998). Results in this study revealed that salt stress did not increase SOD activity in NaCl-unadapted tobacco BY-2 cells, even with exogenous application of proline or betaine. On the other hand, Chen and Dickman (2005) suggest that addition of proline does not increase SOD activity during oxidative stress. These results have good agreement with the results of Demiral and Tu ¨rkan (2004) who found that salt treatment increased SOD activity in both salt-tolerant and saltsensitive rice cultivars but glycinebetaine did not change significantly SOD activity in salt-sensitive rice cultivars under salt stress. Taken together, these results suggest that neither proline nor betaine could enhance SOD activity to scavenge superoxide under salt stress. Salinity accumulates the ROS including H2O2 in plant cells. The metabolism of H2O2 is dependent on various functionally interrelated antioxidant enzymes such as catalases and peroxidases localized in almost all compartments of plant cells. The increase in antioxidant enzyme activities is involved in eliminating H2O2 from salt-stressed roots (Kim et al., 2005). Our results demonstrated that catalase and peroxidase activities in NaCl-unadapted BY-2 cells decreased when the cells were cultured in the NaCl medium. These results are also supported by the findings of Shalata et al. (2001) and Mittova et al. (2004). The reductions in catalase and peroxidase activities suggest that these enzymes were unable to completely detoxify H2O2 generated by salt stress. Under salt stress conditions, catalase (Fig. 4) and peroxidase (Fig. 5) activities increased significantly in the presence of proline. These results are consistent with the results of Khedr et al. (2003) who observed that the activities of catalase and peroxidase decreased under severe salt stress but increased in the presence of proline. Demiral and Tu ¨rkan (2004) found that salt stress decreased the activities of catalase and peroxidase in both saltsensitive and salt-tolerant rice varieties, while salt stress increased the activities of these enzymes in the presence of betaine. However, results obtained in this study show that exogenous betaine increased significantly the activities of catalase but did not increase significantly the activities of peroxidase in BY-2 cells under salt stress. Catalase and
ARTICLE IN PRESS Proline increases antioxidant enzyme activities more than betaine peroxidase play an important role in the fine regulation of ROS concentrations in cells (Elstner, 1987). Proline and betaine have been considered to act as compatible solutes, osmoprotectants and hydroxyl radical scavengers (see Introduction). The present investigation suggests that both exogenous proline and betaine are able to effectively detoxify H2O2 by enhancing the activities of catalase and peroxidase under salt stress. Exogenous proline is more capable than exogenous betaine in enhancing the activities of catalase and peroxidase under salt stress. In this study, a positive relationship among proline, betaine and up-regulation of antioxidant enzyme activities in tobacco BY-2 cells has been observed under salt stress. Therefore, we conclude that up-regulation of antioxidant enzyme activities by proline and betaine scavenges ROS and thereby mitigates the detrimental effects of salt stress. Proline is more effective than betaine in mitigating the detrimental effects of salt stress by upregulation of antioxidant enzyme activities.
Acknowledgments The authors are grateful to Japan Tobacco Inc. for the generous gift of Nicotiana tabacum L. cv. Bright Yellow cultured cells in suspension.
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