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Light dependency of salinity-induced chloroplast damages

Shiro Mitsuya1, Koji Yamane2, Michio Kawasaki3, Mitsutaka Taniguchi4 and Hiroshi Miyake5

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

The contents of Na, K, Cl, chlorophyll and the foliar ultrastructure of rice seedlings grown in NaCl solution at various concentrations were investigated under light and dark conditions. The seedlings were first grown in water for 7 d under a light condition and then in NaCl solutions at various concentrations for 24 h under a light or dark condition. The Na and Cl contents in the 3rd leaves increased as the concentration of NaCl in the culture solution increased, and were significantly higher under a light condition than under a dark condition. The K content was scarcely influenced by the NaCl concentration under both conditions. The chlorophyll content in the 3rd leaves of the seedlings decreased as the NaCl concentrations of the culture solution increased under a light condition but not under a dark condition. In the 3rd leaves of the seedlings grown in the NaCl solution under a light condition, the thylakoids of chloroplasts in mesophyll cells were swollen and showed a wavy configuration. Under a dark condition, however, the thylakoids appeared intact under saline conditions although the leaves accumulated a large amount of Na and Cl than in a light condition. The present study suggests that the damages in the chloroplasts, such as a decrease in the chlorophyll content and the degradation of thylakoids, were caused by a light-dependent reaction and not directly by accumulation of excess salt.

Media summary

In the salt-treated rice plants, the damages in the chloroplasts were caused by a light-dependent reaction and not directly by accumulation of excess salt.

Key Words

Chloroplast, Oxidative reaction, Rice, Salt stress, Ultrastructure

Introduction

Salinity affects 7% of the world’s land surface and is one of the main limiting constraints to global agriculture. In those areas, plant growth was severely affected by salinity through water deficit, salt-specific damages (Munns and Termaat, 1986) or oxidative stress (Hernandez et al., 1995). The salinity-induced decrease in photosynthetic activity (Sakamoto et al., 1998), inhibition of foliar growth (Hu and Schmidhalter, 1998) and ultrastructural changes (Mitsuya et al., 2000) have been reported.

It is important to understand how the plants are damaged by salinity to increase their salt tolerance. Munns and Termaat (1986) concluded that salt injury is due to salt accumulation in transpiring leaves to the levels exceeding the ability of the cells to compartmentalize the salt into the vacuole. On the other hand, Tattini et al. (1995) reported that the salinity-induced decrease of photosynthesis in olive trees was due to the osmotic effect of the salt outside the roots, not to a specific effect of the salt in the leaves. In addition, in our previous study, the damages caused by salinity were not correlated with Na content in the tissue (Mitsuya et al., 2002). Therefore, it is still unclear how the plant cell is affected by salt stress and whether the damage is due to accumulation of excess salt or salt-induced secondary stress.

One of the notable effects of salinity is the degradation of the membranes of cell organelles (Mitsuya et al., 2000). In particular, the thylakoids of the chloroplasts degrade rapidly under salt stress. However, the membranes of etioplasts are scarcely damaged by salinity under dark conditions (Mitsuya et al., 2000). In the present study, to examine whether the degradation of the chloroplasts was caused by accumulation of excess salt, we cultured rice seedlings in solutions containing NaCl at various concentrations under a light or dark condition and examined the contents of some elements (Na, Cl and K) and chlorophyll and the ultrastructure of the 3rd leaves. We show that the degradation of the chloroplasts was caused by a light-dependent oxidative reaction and not directly by accumulation of excess salt.

Methods

Plant material

The seeds of rice (Oryza sativa L. cv. Nipponbare) were surface sterilized with 5% sodium hypochlorite solution for 5 min and thoroughly washed with distilled water. Then the seeds were allowed to imbibe in petri dishes containing distilled water in an incubator kept at about 28℃ until the white coleoptile tips appeared. The uniformly germinated seeds were sown on plastic nets placed on the surface of 300 mL distilled water. After 7 d, the seedlings were transferred to the supporting net on the surface of 300 mL of 0, 0.4, 0.6, 0.8, 1.0 or 1.2 M (light condition) or 0, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2 or 2.4 M NaCl solution (dark condition) in tall beakers (500 mL). Then they were incubated in a growth chamber at 28℃ under a light (under fluorescent white lamps at a light intensity of about 450 μmol m-2 s-1 at the plant level in a 16:8 h light/dark cycle) or dark condition for 24 h.

Measurement of chlorophyll content in the 3rd leaves

After the incubation, the 3rd leaf blades (from bottom) of the seedlings were blotted dry on a filter paper. Chlorophyll was extracted from the leaf blades with 100% ethanol and absorbance of the supernatant was read at 665 and 649 nm using a spectrophotometer (Japan Spectroscopic Co., LTD., Ubest-50). Then the leaf blades were dried at 70 for 48 h and the dry weight was determined. Chlorophyll content was calculated according to the formulae rendered by Knudson et al. (1977) and expressed on a unit-dry-weight basis.

Measurement of element (Na, K, Cl) contents in the 3rd leaves

The 3rd leaf blades of the seedlings were rinsed with distilled water and blotted dry. The dry weight was determined after they were dried at 70 for 48 h and cooled in a desiccation chamber. They were extracted with distilled water at room temperature for 70 h. Contents of Na and K were measured with an atomic absorption spectrometer (Shimadzu Co., Ltd., AA-6400F) in the emission mode. Cl content was measured with a liquid chromatograph (Shimadzu Co., Ltd., LC-10AD). The data were expressed on a unit-dry-weight basis.

Data for chlorophyll content and element contents were statistically analyzed according to Duncan’s multiple range test.

Transmission electron microscopy

For microscopic studies, small samples of the middle part of the 3rd leaf blades grown under light and dark conditions were fixed in Karnovski’s fixative (mixture of 4% paraformaldehyde and 2% glutaraldehyde in 0.05 M phosphate buffer (pH 7.2)) and post-fixed in 2% osmium tetroxide in the same buffer. The samples were dehydrated in a series of graded acetone and propylene oxide and embedded in Spurr’s resin. Ultrathin sections were cut with a diamond knife on an Ultracut-N microtome (Reichert, Nissei) and mounted on grids. Then the sections were stained with uranyl acetate, followed by lead citrate and examined under a transmission electron microscope (Hitachi, H-600). At least 3 samples from each condition were examined with a transmission electron microscope.

Results

In the rice seedlings used in the present experiments, the 3rd leaf was developing at sampling. In the control seedlings, all the leaves were green. In the salt-treated seedlings, the 2nd leaf blades and the tip of the 3rd leaf blades were slightly yellow under the light condition, although all the leaves were green under the dark condition.

Salinity effect on Na, Cl, K and chlorophyll contents

Figure 1 shows the effect of salinity on Na, Cl and K contents in the 3rd leaf blades of the seedlings cultured in a solution containing NaCl at various concentrations under a light or dark condition for 24 h. Under both light and dark conditions, Na and Cl contents increased as the NaCl concentration in the culture solution increased. However, at the same concentration of NaCl in the solution, the leaves under the light condition contained larger amounts of Na and Cl than in the dark condition. Therefore, we observed the ultrastructure of the 3rd leaf blades of the seedlings cultured in 0, 0.6 and 0.8 M NaCl solutions under a light condition and those cultured in 0, 1.8 and 2.4 M NaCl solutions under a dark condition for 24 h. Among these samples, Na and Cl contents were the highest in the seedling cultured in a 2.4 M NaCl solution under a dark condition. Salt treatment under light (except at 0.6 M) and dark conditions did not change the K content significantly.

Figure 2 shows the effect of salinity on the chlorophyll content in the 3rd leaf blades of the seedlings cultured in a solution containing NaCl at various concentrations under a light or dark condition for 24 h. Under the light condition, the chlorophyll content decreased with increasing NaCl concentration, but was not changed under a dark condition.

Figure 1. Na, Cl and K contents in the 3rd leaf blades of the seedlings cultured for 24 h in a solution containing NaCl at various concentrations under a light or dark condition. Data are means SE (n = 5). Vertical bars on the each symbol reprersent SE. (a) Na content. (b) Cl content. Na and Cl contents in the NaCl-treated seedlings were significantly higher than the control (P<0.01). (c) K content. NaCl treatment under light (except at 0.6 M (P<0.05)) and dark conditions did not change the K content significantly.

Figure 2. The relative chlorophyll content (μg / g DW) in the 3rd leaf blades of the seedlings cultured for 24 h in a solution containing NaCl at various concentrations under light and dark conditions (100% = 2.03 0.03 (light), 1.72 0.03 (dark)). Data are means SE (n = 5). Vertical bars on each symbol represent SE. **, significantly different from the control at P<0.01.

Foliar ultrastructure

Figures 3 and 4 show the ultrastructure of the chloroplasts in the mesophyll of the 3rd leaf blades of the seedlings cultured in 0 (control) and 0.8 M NaCl solutions under a light condition, respectively. The chloroplasts of the control seedlings showed no structural distortion and possessed typical well-developed grana and stroma thylakoids (Fig. 3). In the mesophyll of salt-treated seedling, however, the thylakoids of the chloroplasts were swollen (Fig. 4, arrow) and had a wavy shape. Figures 5 and 6 show the ultrastructure of the chloroplasts in mesophyll of the 3rd leaf blades of the seedlings cultured in 0 (control) and 1.8 M NaCl solutions under a dark condition, respectiveliy. The chloroplasts of the control and salt-treated seedlings showed no structural distortion and possessed typical well-developed grana and stroma thylakoids without swelling although the leaves contained a larger amount of Na and Cl than in a light condition.

Fig. 3 to 6. Ultrastructure of the chloroplasts in mesophyll of the 3rd leaf blades of rice seedlings cultured in an NaCl solution under light and dark conditions. Fig. 3 and 5: Control under light and dark conditions, respectively. Fig. 4: Seedlings cultured in 0.8 M NaCl solutions under a light condition. Fig. 6: Seedlings cultured in 1.8 M NaCl solutions under a dark condition. Bar = 1 μm. G, grana thylakoids; St, stroma thylakoids; arrows show swollen thylakoids.

Conclusion

In the salt-stressed leaves under light condition, the chlorophyll content decreased and the thylakoids of the chloroplasts were swollen and showed a wavy shape. It is reported that the structural change and swelling of thylakoid might be due to a change in the ionic composition of the stroma (Salama et al. 1994). However, under the dark condition, the thylakoids of the chloroplasts appeared intact even when the leaf tissues accumulated a larger amount of Na and Cl than under the light condition. These results indicated that the degeneration of the thylakoids of the chloroplasts caused by salinity was not directly caused by accumulation of salt to the excessive level in the tissue. The chloroplast is an important intracellular light-dependent generator of activated oxygens. In addition, the change of the thylakoids has been reported as a typical symptom of oxidative stress (Hernandez et al., 1995). This suggested that the damages in chloroplasts were induced by a photooxidative reaction caused by salt stress and not directly correlated with salt content in the tissue.

References

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