The seeds of rice (Oryza sative. Jinxing1) were sterilized by H₂O₂ (10%) for 30 min and washed with deionized water, then soaked in water for 6 h and germinated in an incubator at 28˚C. When the length of hypocotyl was about 1 cm, seedlings were transplanted to plastic pots (diameter 10 cm) filled with deionized water in an incu- bator with the illumination of 6 klx (12 h light per day) at 28˚C. When the second leaves appeared, the seedlings were cultured in a 1/2 Hoagland nutrient solution. The nutrient solution was renewed every three days to stabil- ize pH. When the third leaves appeared, the seedlings were separately treated with different concentration of Ce⁴⁺ (0.5, 5, 10 mg/L) supplied as Ce(NH₄)₂(NO₃)₆. The control was treated with same amount of deionized water. Samples were collected for analyses at a predetermined time.

Seedlings viability was assayed using 2,3,5-triphenyltetrazolium chloride (TTC) following the method of Iborra et al. [13] . Following Ce⁴⁺ treatment, the TTC solution was infiltrated into the rice cells. This assay directly de- termines the mitochondrial electron transport activity. The relative level of TTC reduction to formazan quanti- fies the cell viability by the spectrophotometric assay of the red formazan [13] . Fresh seedlings (200 mg) were collected in a tube and 8 ml TTC solution (0.8% TTC in 0.05 M phosphate buffer, pH 7.4, and 0.5 mL/L Tween 20) were added and the mixture was infiltrated for 5 min at reduced pressure. The seedlings samples were incu- bated in the TTC solution for 24 h at 25˚C in the dark, then seedlings were removed and rinsed with distilled water, placed in separate quartz tubes containing 3 ml of 95% ethanol in each, and submerged for 24 h at 25˚C in the dark. The absorbance was measured at 485 nm. Cell viability was expressed as the absorbance of the sample at 485 nm per gram of biomass (fresh wt.).

pH measurement was performed by the pH meter (Radiometer Copenhagen, PHM210, Tacussel, France).

Rice seedlings were subjected to the same concentrations of Ce⁴⁺ as in the electrolyte leakage assay. The elec- trolyte leakage was quantified by electrical conductivity measurement. Membrane stability was expressed in terms of the electrolytic conductivity [14] . This method measures the increased electrolyte diffusion induced by Ce⁴⁺ as a result of the changed cell membrane permeability. A total of 200 mg seedlings were transferred from the Ce⁴⁺-treated samples to a 10 ml centrifuge tube containing 4 ml ultrapure water at predetermined time of cultivation. Absolute electrical conductivity (uS cm⁻¹) was measured at 25˚C using a Model DDS-307 conduc- tivity meter.

Malonyl dialdehyde (MDA), a final product of lipid peroxidation, was measured to evaluate the extent of lipid peroxidation in seedling hydroponic culture of rice. The MDA was measured by thiobarbituric acid (TBA) reac- tion [15] . Briefly, seedlings (200 mg) were homogenized in 4 ml 0.1% trichloroacetic acid (TCA). The homo- genate was centrifuged at 13,000 g for 10 min. To 2 ml aliquot of the supernatant was added 4 ml 20% TCA containing 0.5% TBA. The mixture was heated at 95˚C for 30 min and quickly cooled down in an ice bath. After centrifugation at 10,000 g for 10 min, the absorbance of the supernatant was measured at 532 nm and the unspe- cific turbidity was exempted by subtracting the absorbance of the same sample at 600 nm. MDA concentration was calculated using the molar extinction coefficient of 155 mM⁻¹·cm⁻¹.

Cell membrane permeability was analyzed using Evans blue dye following the method of Suzuki et al. [16] with a slight modification. Seedlings (200 mg) washed three times with 50 mM phosphate buffer (pH 5.8); seedlings were then placed on the sterilized dry filter paper. After doing so, the seedlings were collected in a tube and stained for 5 min with a 0.15% (w/v) solution of Evans Blue. Afterwards, the seedlings were re-filtered and washed five times with PBS to remove the excess dyes. The stained seedlings were collected and re-suspended in 5 ml solution consisting of 1.0% (w/v) SDS and 50% (v/v) methanol for 30 min at 50˚C. The solution was then cooled down to room temperature and the supernatant was collected and its absorbance at 600 nm was measured.

Figure 1 showed that the seedlings cell viability was hardly affected during the whole period when the concen- tration of Ce⁴⁺ was below 0.5 mg/L. However, the cell viability began to decreased rapidly after 2 days in the case of 5 mg/L Ce⁴⁺ and decreased 49.5% in the case of rice of that of control on Day 6. Further increase of Ce⁴⁺ (above 10 mg/L) reduced the cell viability, while the cell viability decreased 65.2% in the case of rice on day 8 after the addition of the Ce⁴⁺.

Figure 2 displays the time course of the conductivity with the addition of Ce⁴⁺. The conductivity was hardly affected compared to that of the control culture when the Ce⁴⁺ content was 0.5 mg/L. When the Ce⁴⁺ content was raised to 10 mg/L, however, the conductivity began to increase rapidly after 2 days and increased 693.3% in the case of rice of that of the control on Day 4, indicating that a net outflow of ions from the treated cells.

To further confirm the changes in cell membrane permeability caused by the Ce⁴⁺ addition, the peroxide product of the main membrane lipids as a marker of membrane lipid breakage, the production of MDA, was tested. Fig- ure 3 showed that MDA was induced by Ce⁴⁺. When the content of Ce⁴⁺ was 0.5 mg/L, the accumulation of MDA was slight. However, the MDA content increased 123.1% of that of the control on Day 5 after the addition of 10 mg/L Ce⁴⁺ and reached 158.3% in the case of rice on Day 7.

Figure 4 shows the dynamic changes of medium pH induced by Ce⁴⁺. As shown in Figure 4, upon the addition of Ce⁴⁺ the extra-cellular medium pH sharply decreased to a maximum value then began to increase and gradu- ally restored to its initial level in the case of rice. The increase in Ce⁴⁺ content led to a severer acidification of the extra-cellular medium. For example, when Ce⁴⁺ concentration was 0.5 mg/L, no obvious change in extra- cellular pH was observed. However, when Ce⁴⁺ concentration was raised to 10 mg/L, the extra-cellular pH de- creased 1.0 unit compared to that of the control sample. At Day 2, all culture media returned to its initial level. These results indicated that Ce⁴⁺ might take a function of inhibiting proton influx into cells across plasma mem- brane.

Integrated cell membrane is impermeable for macromolecules such as Evans blue. When cell membrane under- goes an injury or loses its selective permeability, macromolecules can penetrate it. Thus, the amount of Evans blue entering and binding on cells reflects the degree of loss of cell membrane integrity. The change in cell membrane permeability was also reflected by the Evans blue assay. When the content of Ce⁴⁺ was 0.5 mg/L, it was evident from Figure 5 that the cell membrane integrity was well maintained as normal. However, when the content of Ce⁴⁺ was higher than 5 mg/L, an obvious loss in cell membrane integrity was observed. These results clearly indicated that Ce⁴⁺ broke the membrane integrity of rice cells when their content reached 5 mg/L or more.