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

Abstract

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⁺.

Introduction

The rare earth elements (REEs), which have the similar chemical properties, include a group of 17 trivalent metallic elements. In China, REEs enriched fertilizers have been used since the 1970s¹. REEs enter into the mesophyll cells via apoplast and symplast channels or plasmodesmata^2,3. It has been observed that the appropriate amount of REEs has positive effects on seed germination, harvest quality, chlorophyll content, photosynthetic rate, plant biomass and root development^4,5,6. The growth of tobacco seedlings and light reactions of photosynthesis were stimulated by the suitable concentration of trivalent lanthanum (La³⁺)⁷. However, the results of field trials and laboratory studies on La³⁺ are still contradictory^8,9,10.

Reactive oxygen species (ROS) mainly include hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂), superoxide anion and hydroxyl radical (OH^·). In the plant cells, ROS could act as a signal molecule and trigger a series of cellular responses^11,12. It is known that ROS are toxic to plants if the accumulation levels of ROS exceed the detoxification capacity of the plant tissues. However, there are various mechanisms to cope with oxidative damage in plants. The enzymatic antioxidants, including superoxide dismutase (SOD, EC.1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), glutathione-S-transferase (GST, EC. 2.5.1.18), catalase (CAT, EC.1.11.1.6) and glutathione reductase (GR, EC 1.6.4.2), are involved in protecting plant cells from oxidative damage by scavenging ROS^13,14,15.

Hormesis is defined as a phenomenon that low doses of an otherwise harmful agent could result in stimulatory or beneficial effects¹⁶. Accurate description of hormetic dose-response is important for the determination of the efficacy and hazards of La³⁺. In this study, the instantaneous change of ROS was investigated in the root cells treated by La³⁺ with the laser-scanning confocal microscopy. Furthermore, the hormetic effects of La³⁺ on the defense system were investigated in the rice seedlings of Oryza sativa L. (cv Shengdao 16).

Results

Effect of La³⁺ on the levels of MDA, H₂O₂, and soluble protein

The level of MDA and H₂O₂ was significantly increased by increasing solution La concentrations from 0.5 to 1.5 mM (Fig. 1A,B), while the level of was significantly increased by 1.0 and 1.5 mM La³⁺ (Fig. 1C). However, the level of H₂O₂ and was not significantly decreased by 0.05 and 0.1 mM La³⁺ (Fig. 1B,C). Moreover, The soluble protein content was significantly decreased by 0.05 and 0.1 mM La³⁺ (Fig. 1D), but no effects was observed by increasing solution La concentrations from 0.5 to 1.5 mM (Fig. 1D).

Figure 1

Effect of La³⁺on MDA, H₂O₂, and soluble protein level in the root of rice.

(A) Effect of La³⁺ on the level of MDA. (B) Effect of La³⁺ on the H₂O₂. (C) Effect of La³⁺ on the level of . (D) Effect of La³⁺ on the level of soluble protein. Values represent means ± SE (n = 3). Different letters indicate significant differences (P < 0.05) according to tukey’s test.

Effect of La on the instantaneous change of ROS in the root cells

The fluorescence of ROS (green) 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 Software. Compared to the control, the level of ROS was declined in the root cells treated with 0.05 mM La³⁺ (Figs 3,4 and 5).

Figure 2

The regions of interest (ROI 1–3) in the root treated with MES-KCl buffer or 0.05 mM La³⁺.

The root cells were labeled with H₂DCF-DA and ROS (green) were imaged. The regions of interest (ROI 1–3) were circled with Leica Confocal Software. (A) The regions of interest (ROI 1–3) in the root treated with MES-KCl buffer. (B) The regions of interest (ROI 1–3) in the root treated with 0.05 mM La³⁺. Bar: 75 μm.

Figure 3

Effects of MES-KCl buffer on ROS in the root cells.

The root cells were labeled with H₂DCF-DA and ROS (green) were imaged. The laser-scanning mode was XYT and the time interval between two sections was 10.0 s. Thirty optical sections were acquired with the laser-scanning confocal microscope and the MES-KCl buffer was added at about 40 s. Ten images at different time were selected in the figures. Bar: 75 μm.

Figure 4

Effects of 0.05 mM La³⁺ on ROS in the root cells.

The root cells were labeled with H₂DCF-DA and ROS (green) were imaged. The laser-scanning mode was XYT and the time interval between two sections was 10.0 s. Thirty optical sections were acquired with the laser-scanning confocal microscope and 0.05 mM La³⁺ was added at about 40 s. Ten images at different time were selected in the figures. Bar: 75 μm.

Figure 5

Effect of MES-KCl buffer or 0.05 mM La³⁺ on ROS in the root cells.

(A) Effect of MES-KCl buffer on ROS in the root cells. (B) Effects of 0.05 mM La³⁺ on ROS in the root cells. The arrow indicates the time that MES-KCl buffer or 0.05 mM La³⁺ was added at about 40 s. Values represent means ± SE (n = 3).

Effect of La³⁺ on SOD, POD and CAT activity and La³⁺ accumulation

Antioxidant assays were performed on the roots of seedlings collected after 13 days treatment with La³⁺. Compared to the control, the activity of SOD was significantly increased in roots after La³⁺ treatments (Fig. 6A). The activity of POD and CAT was significantly increased by 0.05 and 0.1 mM La³⁺ (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 La³⁺ (Fig. 6A,B), but the highest CAT activity was at 0.05 mM La³⁺ (Fig. 6C). Moreover, La³⁺ particles were located in the cell wall with the technique of transmission electron microscope (Fig. 7).

Figure 6

Effect of La³⁺ on the activity of SOD, POD and CAT in the root of rice.

(A) Effect of La³⁺ on the activity of SOD. (B) Effect of La³⁺ on the activity of POD. (C) Effect of La³⁺ on the activity of CAT. Values represent means ± SE (n = 3). Different letters indicate significant differences (P < 0.05) according to tukey’s test.

Figure 7

The subcellular distribution of La in the root.

(A) Control, showing no La particle deposited in the cell wall (CW); (B) root cell treated with 1.0 mM La³⁺, showing La particles located in the CW. The arrow indicates La particles.

Discussion

In this study, the level of H₂O₂, and MDA was significantly induced by 1.0 and 1.5 mM La³⁺, but not significantly inhibited by 0.05 and 0.1 mM La³⁺. It has been reported that low concentrations of La³⁺ alleviated the oxidative damage induced by UV-B radiation through inhibiting the production of H₂O₂, and MDA¹⁷. The results of Wang et al. reported that La³⁺ protected soybean plants from oxidative stress by improving the defense system of plants¹⁸. However, the level of H₂O₂, and MDA was not significantly affected by low concentrations of La³⁺ in this study. The reason may be that the concentrations of La³⁺ were different from the previous studies. 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 stimulation¹⁹. In this study, it was found that the activity of CAT and POD was induced by 0.05 and 1.0 mM La³⁺, but not affected by 0.5, 1.0 and 1.5 mM La³⁺. Additionally, La³⁺ 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 La³⁺. The formation of H₂O₂ induced by higher concentrations of La³⁺ may be associated with the increased activity of SOD for conversion.

The concentration of REEs is not consistent in the previous studies. The results of Ippolito et al. showed that 5.0 mM La³⁺ did not cause either visible symptoms on plants or significant effects on ROS production, chlorophyll content and lipid peroxidation in common duckweed²⁰. Diatloff et al. found that La³⁺ induced the growth of corn and mungbean when the concentration was below 0.2 μM⁹. However, Chen et al. reported 60 mM La³⁺ significantly promoted the growth of callus²¹. In this study, it appears that the higher concentration of La³⁺ induces oxidative stress in the root cells and the appropriate concentration of La³⁺ 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 cells²². 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 La³⁺, which leads to the different ROS distribution in the root cells. Wang et al. reported that La³⁺ protected soybean plants from oxidative stress by directly reacting with ROS¹⁸. Our results also show that the appropriate concentration of La³⁺ could decrease the level of ROS with confocal microscopy.

In this study, it was found that the effect of La³⁺ on the antioxidant metabolism was related to the concentration of La³⁺. Once the accumulation of La³⁺ exceeds the detoxification capacity of the plant tissues, it will be toxic to plant cells²³. In the study, this threshold was reached at 0.5 mM La³⁺ 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 La³⁺.

In summary, the probe H₂DCF-DA was used to investigate the instantaneous change of ROS with the laser-scanning confocal microscopy. It showed that the appropriate concentration of La³⁺ 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 La³⁺ (0.05, 0.1, 0.5, 1.0 and 1.5 mM).

Methods

Plant material and plant growth

Seeds of rice (O. sativa L. cv Shengdao 16) were sterilized by soaking in 75% alcohol for 1 min, in 0.1% mercury chloride for 15 min and in 1.0% sodium hypochlorite for 20 min. Then the seeds were rinsed five times with sterilized water and germinated in half-strength Murashige and Skoog agar medium at pH 5.8 (0.75 mM MgSO₄, 10 mM NH₄NO₃, 9.4 mM KNO₃, 0.625mM KCl, 1.5 mM CaCl₂, 2.5 μM KI, 50 μM H₃BO₃, 50 μM FeSO₄, 50 μM MnSO₄, 15 μM ZnSO₄, 0.05 μM CuSO₄, 0.05 μM CoCl₂, 0.5 μM Na₂MoO₄, 50 μM Na₂H₂EDTA, 0.15 μM thiamine, 1.2 μM pyridoxine, 2 μM nicotinic acid, 275 μM inositol, 0.56% agar, 3.0% sucrose, 0.05% Mes). Subsequently, La(NO₃)₃ was added to the basal medium before autoclaving to obtain La³⁺ concentrations of 0, 0.05, 0.1, 0.5, 1.0 and 1.5 mM. Plants were grown 13 days in the different concentration of La³⁺ at 25.0 ± 2 °C using a 14/10 h light/dark cycle under a light intensity of 200 μmol m⁻² s⁻¹ in a growth chamber.

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 formation²⁵. The CAT activity was assayed at 405 nm based on the principle that H₂O₂ could react with ammonium molybdate and form a stable complex and one unit of CAT decomposes 1.0 μM H₂O₂ per minute²⁶. The POD activity was determined by an increase in absorbance at 470 nm during the oxidation of guaiacol²⁷.

Assay the levels of soluble protein, H₂O₂, and MDA

The plant samples were obtained as the assay of antioxidant enzyme activities. The same supernatant was used for the determination of soluble proteins, H₂O₂, and malondialdehyde (MDA). The content of H₂O₂ was determined at 405 nm based on the principle that H₂O₂ could form a stable complex with ammonium molybdate²⁶. The soluble proteins were assayed with bovine serum albomin as standard protein according to the Bradford method²⁸. The level of was determined at 530 nm according to procedures described by (Wang and Luo)²⁹ and the level of MDA was measured for it could react with thiobarbituric acid³⁰.

Effect of La on the instantaneous change of ROS in the root cells

The production of ROS in the roots was visualized with 2′,7′-dichlorofluorescein diacetate (H₂DCF-DA, Molecular Probes). For assessment of ROS, the rice roots were immersed in 1.5 ml of 10 μM DCFH-DA (dissolved in MES-KCl buffer, KCl 50 mM, MES 10 mM, pH 5.5) for 2 hr at 20 °C. Then the roots were rinsed with MES-KCl buffer (KCl 50 mM, MES 10 mM, pH 5.5) to remove DCFH-DA solution from the surface of roots.

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 La³⁺ 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.

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).