Lanthanum regulates the reactive oxygen species in the roots of rice seedlings

In this study, the effects of La3+ on the reactive oxygen species (ROS) and antioxidant metabolism were studied in the roots of rice (Oryza sativa L. cv Shengdao 16) exposed to increasing concentrations of La3+ (0.05, 0.1, 0.5, 1.0, and 1.5 mM). The level of hydrogen peroxide, superoxide anion, and malondialdehyde was increased by 0.5, 1.0 and 1.5 mM La3+, and the activity of catalase and peroxidase was increased by 0.05 and 0.1 mM La3+. However, La3+ treatments stimulated superoxide dismutase activity in the roots of rice seedlings at all tested concentrations. In addition, the probe 2′,7′-dichlorofluorescein diacetate (H2DCF-DA) was used to investigate the instantaneous change of ROS in the root cells with the laser-scanning confocal microscopy. The result indicated that ROS level was declined after treated with 0.05 mM La3+. The results showed that the appropriate concentration of La3+ decreased the level of ROS, and hormetic effects on the antioxidant metabolism were found in the roots of rice exposed to 0.05, 0.1, 0.5, 1.0, and 1.5 mM La3+.

was imaged in the cells of root and the regions of interest (ROI 1-3) were circled with Leica Confocal Software (Fig. 2). The fluorescence intensity of ROS in the root cells was processed and quantified with Leica Confocal  Effect of La 3+ on SOD, POD and CAT activity, and La 3+ accumulation. Antioxidant assays were performed on the roots of seedlings collected after 13 days treatment with La 3+ . Compared to the control, the activity of SOD was significantly increased in roots after La 3+ treatments (Fig. 6A). The activity of POD and CAT was significantly increased by 0.05 and 0.1 mM La 3+ (Fig. 6B), However, POD and CAT activity was unaffected by increasing solution La concentrations from 0.5 to 1.5 mM (Fig. 6B). In addition, the highest increase of SOD and POD activity was observed at 0.1 mM La 3+ (Fig. 6A,B), but the highest CAT activity was at 0.05 mM La 3+ (Fig. 6C). Moreover, La 3+ particles were located in the cell wall with the technique of transmission electron microscope (Fig. 7).   In addition, the increase of antioxidants induced by lanthanum nitrate treatment at low concentrations has been reported in the aged Oryza sativa L., and the production of ROS was successfully controlled by the antioxidant stimulation 19 . In this study, it was found that the activity of CAT and POD was induced by 0.05 and 1.0 mM La 3+ , but not affected by 0.5, 1.0, and 1.5 mM La 3+ . Additionally, La 3+ treatments stimulated SOD activity in the roots of rice seedlings at all tested concentrations. The results indicated that some protective enzymes were activated by 0.05 and 1.0 mM La 3+ . The formation of H 2 O 2 induced by higher concentrations of La 3+ may be associated with the increased activity of SOD for ⋅ − O 2 conversion. The concentration of REEs is not consistent in the previous studies. The results of Ippolito et al. showed that 5.0 mM La 3+ did not cause either visible symptoms on plants or significant effects on ROS production, chlorophyll content, and lipid peroxidation in common duckweed 20 . Diatloff et al. found that La 3+ induced the growth of corn and mungbean when the concentration was below 0.2 μ M 9 . However, Chen et al. reported 60 mM La 3+ significantly promoted the growth of callus 21 . In this study, it appears that the higher concentration of La 3+ induces oxidative stress in the root cells, and the appropriate concentration of La 3+ on growth may be related with the different plant species.
REEs can be absorbed into plant cells, which is the basis for interpreting biochemical effects of REEs on plant cells 22 . In this study, the distribution of ROS is not consistent in the root cells. There are more ROS in some root cells, and less ROS in the other root cells. The reason may be that the root cells accumulate the different amount of La 3+ , which leads to the different ROS distribution in the root cells. Wang et al. reported that La 3+ protected soybean plants from oxidative stress by directly reacting with ROS 18 . Our results also show that the appropriate concentration of La 3+ could decrease the level of ROS with confocal microscopy.
In this study, it was found that the effect of La 3+ on the antioxidant metabolism was related to the concentration of La 3+ . Once the accumulation of La 3+ exceeds the detoxification capacity of the plant tissues, it will be toxic to plant cells 23 . In the study, this threshold was reached at 0.5 mM La 3+ in the nutrient solution. Hormetic effects generally show two kinds of trends, including the low-dose-stimulation and the high-dose-inhibition effects 16,24 . In this study, the hormetic effects on the antioxidant metabolism were also observed in the roots of rice treated with low and high concentrations of La 3+ .
In summary, the probe H 2 DCF-DA was used to investigate the instantaneous change of ROS with the laser-scanning confocal microscopy. It showed that the appropriate concentration of La 3+ decreased the level of ROS in the root cells. Hormetic effects on the antioxidant metabolism were found in the roots of rice exposed to increasing concentrations of La 3+ (0.05, 0.1, 0.5, 1.0, and 1.5 mM). Assay antioxidant enzyme activities. Plants were collected and the fresh weight (FW) of roots was determined in 13 day-old seedlings. The roots biomass (0.5 g in fresh mass) was homogenized under ice-cold conditions in 5.0 ml of extraction buffer containing 50 mM phosphate buffer (pH 7.5), 1.0% polyvinylpyrrolidone (PVP), 0.5% Triton X-100 and 1 mM EDTA, centrifuged at 10,000 × g at 4 °C for 20 min to remove particulate plant debris. The supernatant was used for the assay of antioxidant enzyme activities. The SOD activity was determined at 550 nm, and one unit of SOD activity was the amount of enzyme that inhibits 50% nitrite formation 25 . The CAT activity was assayed at 405 nm based on the principle that H 2 O 2 could react with ammonium molybdate and form a stable complex, and one unit of CAT decomposes 1.0 μ M H 2 O 2 per minute 26 . The POD activity was determined by an increase in absorbance at 470 nm during the oxidation of guaiacol 27 . A laser-scanning confocal microscope (Leica TCS SP2, Germany) with an argon-ion laser as the excitation source at 488 nm was used to view the sites of ROS in the root cells. The laser-scanning mode was XYt, 30 optical sections were acquired, and the time interval between two sections was 10.0 s. To investigate the instantaneous change of ROS in the root, MES-KCl buffer or 0.05 mM La 3+ was added into 35 mM petri dish after four optical sections were scanned, respectively. Then the regions of interest (ROI 1-3) were circled with Leica Confocal Software, and the fluorescence intensity of ROI was acquired. Data were processed with a Leica TCS Image Browser, and transferred to Adobe Photoshop 6.0 for preparation of figures.

Plant material and plant growth.
Transmission electron microscope analysis. The roots were fixed for 2 h at 4 °C in 2.5% (v/v) glutaraldehyde and 0.1 M phosphate buffer solution (pH 7.3), and postfixed in 1% (w/v) aqueous osmium tetraoxide for 2 h. Samples were dehydrated in a 50-100% ethanol series and embedded in Epon 812 resin. Ultra-thin sections of 70 nm thickness were cut with an Ultracut Eultramicrotome (Leica, Germany) and stained with uranyl acetate and lead citrate. Then the subcellular distribution of La was detected with a Hitachi H-600 transmission electron microscope (TEM).

Statistical analyses.
The assays for oxidative stress and the soluble protein were carried out in three different experiments, and results are expressed as mean ± standard error (SE). Statistical comparisons were done with one-way ANOVA using SPSS 16.0 for Windows (SPSS Inc., Chicago, USA). Tukey test was performed for post hoc comparisons when the difference was significant (P < 0.05).