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Salinity-induced chloroplast damages in rice leaves (Oryza sativa L.) are reduced by pretreatment with methyl viologen

Koji Yamane1, Shiro Mitsuya2, Michio Kawasaki3, Mitsutaka Taniguchi4 and Hiroshi Miyake5

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
1
Email i031014d@mbox.nagoya-u.ac.jp
2
Email mitsuya@agr.nagoya-u.ac.jp
3
Email kawasaki@agr.nagoya-u.ac.jp
4
Email taniguti@agr.nagoya-u.ac.jp
5
Email miyake@agr.nagoya-u.ac.jp

Abstract

We investigated the effects of pretreatment with methyl viologen (MV) on the salinity-induced chloroplast degeneration in rice seedlings. After the seedlings were grown in a hydroponic nutrient solution for 3 weeks, they were treated with 100 nM MV mixed in the hydroponic culture for 3 days, and then they were subjected to 200 mM NaCl for 3 days without MV. In the plants without MV pretreatment, the chlorophyll content drastically decreased, and the swelling of thylakoids and destruction of thylakoid membranes were observed under 200 mM NaCl. On the other hand, these damages were suppressed in the plants pretreated with MV. The activities of CuZn-SOD and Fe-SOD, which localize in chloroplasts, increased under salt stress in both plants with and without MV pretreatment. In the plants under salt stress without MV pretreatment, ascorbate peroxidase (APX) activity did not differ from that of control. However, in MV-pretreated plants, APX activity was about 1.2- to 1.3-fold higher than control under salt stress. These results suggest that MV increases salt tolerance in chloroplasts by increasing APX activity.

Media summary

Pretreatment with methyl viologen suppressed reduction of chlorophyll content and ultrastructural changes of chloroplasts under salinity by increasing APX activity under salinity.

Key Words

Ascorbate peroxidase, Methyl viologen, Salt stress, Superoxide dismutase, Thylakoid, Ultrastructure

Introduction

When plants are affected by salt stress, prominent swelling of thylakoids, which is a typical symptom of oxidative damage (Hernndez et al. 1995), is induced at the early stage of the damage (Yamane et al. 2003). Therefore, suppression of the oxidative damage of chloroplasts is necessary to reduce the adverse effects of salinity. Though high concentrations of active oxygen species (AOS) in cells and organelles can lead to oxidative damage, there are some recent reports indicating that AOS are not simply toxic by-products of metabolism but they function as signaling molecules (Vam Camp et al. 1998). Methyl viologen (MV), also known as paraquat, is a herbicide widely used in agriculture. It has long been known to exert its toxic effects by catalyzing the transfer of electrons from photosystemⅠto oxygen, which produces O2- (Cha and McRae 1982). In the present study, we report that pretreatment with low level of MV can suppress the ultrastructural damages in chloroplasts under salt stress.

Methods

Plant materials and stress treatment

Seeds (Oryza sativa L. cv. Nipponbare) were sown on hydroponic culture containing nutrients according to Mae and Ohira (1981) and grown in a growth chamber under 12 h photoperiod at 400-500μmol m-2s-1 and 28/25℃ (day/night) for 3 weeks. Three-week-old plants were treated with MV mixed in the hydroponic culture for 3 days, and then the plants were subjected to 200 mM NaCl for 3 days without MV.

Measurement of chlorophyll content

Chlorophyll content of fully expanded uppermost leaves (5th leaves) was determined in 100 % ethanol according to Knudson et al. (1977).

Enzyme extraction and assays

SOD activity was assayed according to Beyer and Fridovich (1987). Activities of different forms of SOD were identified according to Yu and Rengel (1999). The activity of APX was measured following Nakano and Asada (1981).

Electron microscopy

Small segments of leaves were fixed according to Yamane et al. (2003). Ultrathin sections (70-90 nm in thickness) were cut with a diamond knife and placed on 150 mesh copper grid. The grids were stained with 2% uranyl acetate for 25 min followed by lead citrate for 5 min. Then the sections were examined on a Hitachi H600 transmission electron microscope at 100 kV.

Results

Changes in chlorophyll content and ultrastructure

When the plants were subjected to NaCl, immediate reduction of chlorophyll content was observed. On the other hand, pretreatment with 100 nM MV for 3 days suppressed the reduction of chlorophyll content (Fig. 1). The chloroplast possessed well-developed granal and stromal thylakoids (Fig. 2). Treatment with 100 nM MV alone did not induce any ultrastructural changes (Fig. 3). After treatment for 2 days, thylakoids swelled (Fig. 4A). NaCl-treatment for 3 days caused severe damage. On the other hand, in the chloroplasts pretreated with MV, the distortion of thylakoid membrane was effectively suppressed under NaCl for 2 days (Fig. 5A). Though the arrangement of thylakoids was slightly disturbed under NaCl for 3 days, severe damage was suppressed by the pretreatment with MV (Fig. 5B).

Figure 2. Ultrastructure of a chloroplast in a plant grown for 3 weeks before NaCl treatment.
Figure 3. Ultrastructure of a chloroplast in a plant treated with MV for 3 days.
Figure 4. Ultrastructure of a chloroplast in plants treated with 200 mM NaCl (A: for 2 days, B for 3 days).
Figure 5. Ultrastructure of a chloroplast in plants treated with 200 mM NaCl (A: for 2 days, B for 3 days) after treatment with 100 nM MV for 3 days.

Figure 1. Changes in chlorophyll content during growth for 3 days under 200 mM NaCl with and without MV pretreatment. Data are means SE (n=6). ** indicates significant differences from control at P<0.01 (Duncan's multiple range test).

Changes in antioxidant enzyme activity

Changes in CuZn-SOD and Fe-SOD activities during growth for 3 days under 200 mM NaCl with and without MV pretreatment are shown in Figure 6A and B. After the plants without MV pretreatment were exposed to 200 mM NaCl, CuZn-SOD activity increased rapidly and Fe-SOD activity increased gradually. The two SOD activities increased by pretreatment with MV as compared with control at 0 day. The activities of CuZn-SOD and Fe-SOD were enhanced 1.1- and 1.5-fold, respectively. In addition, both SOD activities increased gradually during salt stress in the plants pretreated with MV. The changes in APX activity of the plants with and without MV pretreatment were monitored during growth in 200 mM NaCl for 3 days (Fig. 7). APX activity did not differ from control during the NaCl treatment. In the plants pretreated with MV, APX activity was enhanced about 1.1-fold at 0 day and became higher than control by about 1.2- ~ 1.3-fold during salt stress.

Figure 6. Changes in the activities of CuZn-SOD and Fe-SOD during growth for 3 days under 200 mM NaCl with and without MV pretreatment. Data are meansSE (n=3). * indicates significant differences from control at P<0.05 (Duncan's multiple range test).

Figure 7. Changes in the activities of APX during geowth for 3 days under 200 mM NaCl with and without MV pretreatment. Data are meansSE (n=3). * and ** indicate significant differences from control at P<0.05 (Duncan's multiple range test).

Conclusion

The activities of CuZn-SOD and Fe-SOD, which localize in chloroplasts, increased under salt stress in both plants with and without MV pretreatment. SOD catalyzes the dismutation of O2- to H2O2 and O2. Therefore, in the present study, H2O2 probably generated at higher rates than control in the chloroplasts of the plants under salt stress, because Fe-SOD activity was gradually increased during salt stress. H2O2 which is generated by the reaction of SOD is generally scavenged by APX. However, APX activity of the plants without MV pretreatment was not increased under salt stress. On the other hand, APX activity of the plants pretreated with MV was generally higher than the control. It is suggested that in the plants pretreated with MV, the concentration of H2O2 in chloroplasts was lower than that of the plants without MV pretreatment under salt stress due to a higher APX activity. Therefore, the reduction of chlorophyll content and ultrastructural changes seem to be suppressed by MV pretreatment.

References

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Mae T and Ohira K (1981). The remobilization of nitrogen related to leaf growth and senescence in rice plants (Oryza sativa L.). Plant and Cell Physiology 22, 1067-1074.

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Yu Q and Rengel Z (1999). Drought and salinity differentially influence activities of superoxide dismutases in narrow-leafed lupins. Plant Science 142, 1-11.

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