Effects of Rare Earth Lanthanum and Cerium on Antioxidant Enzyme Activities in Soybean Leaves

Ren Hong-yu, Miao Yan-li, Wang Zhen-hua, Zhuang Zhong, Li Xin, and Zhang Xing-wen

1 College of Resources and Environmental Sciences, Northeast Agricultural University, Harbin 150030, China

2 College of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001, China

Abstract: Rare earth has a long history of agricultural use and can improve the yield and quality of agricultural by-products,but its regulation mechanism is still unclear. The aim of the study was to investigate the effects of lanthanum and cerium on the activities of peroxidase, catalase and ascorbate peroxidase in leaves of soybeans [Glycine max (L.) Merr]. Soybean varieties of Dongnong 42, Dongnong 47 and Dongnong 52 from Northeast China were used as experimental materials. The samples were tested by spraying different concentrations of LaCl3 (120, 150 and 180 mg · L-1), CeCl3 (30, 60 and 90 mg · L-1) and LaCl3+CeCl3 (30, 40 and 60 mg · L-1 mix) solutions on the leaves at the seedling stage and the early fl owering stage of soybeans. The results showed that the suitable species and concentrations of lanthanum and cerium could increased the activities of antioxidant enzymes in soybean leaves, but the effects of lanthanum and cerium on different varieties of soybeans were also different. For Dongnong 42, the optimum lanthanum and cerium treatment was 60 mg · L-1 LaCl3+CeCl3, and the activities of peroxidase, catalase and ascorbate peroxidase increased by 11.36% (p＜0.05),13.89% (p＞0.05) and 50.00% (p＜0.05) compared with those of CK. The optimal lanthanum and cerium treatment of Dongnong 47 was 40 mg · L-1 LaCl3+CeCl3. Compared with those of CK, the above three enzyme activities increased by 15.80% (p＜0.05), 51.52% (p＜0.05)and 20.75% (p＞0.05). The optimal lanthanum and cerium treatment of Dongnong 52 was 60 mg · L-1 LaCl3+CeCl3, which increased the activities of the three enzymes by 33.49% (p＜0.05), 19.23% (p＞0.05) and 23.53% (p＜0.05) compared with those of CK.

Key words: lanthanum, cerium, soybean, antioxidant enzyme

Introduction

Rare earth is a general term of 17 elements in the periodic table, including lanthanum (La) and cerium(Ce), together with their families of scandium (Sc)and yttrium (Y). In 2017, the world's rare earth production was about 178 200 tons, while China's rare earth production was 160 000 tons, accounting for 89.88%. By far, China is still the country with the most abundant rare earth resources, the most complete variety and high grade. China has the largest supply of products and dominates the global rare earth market(Liu, 2018). Over the decades, rare earth has been widely used in agriculture, animal husbandry, industry,medical treatment and many other fields. Studies have shown that within the appropriate concentration range,rare earth can not only promote seed germination and growth (Wu and Feng, 2012), but also improve the photosynthetic organs and activities of plants.Rare earth also can effectively improve the activities of enzymes in plants (Xu and Wu, 2006), promote the physiological metabolism of plants, and thereby enhance the ability of plants to resist stress (Xuet al., 2010). Zhanget al.(2017) found that 0.08 mm lanthanum and acid rain (pH 4.5) combined treatment can promote nitrogen assimilation in soybean seedling roots, not only to solve the damage of soybean seedlings caused by acid rain, but also promote the growth of soybean.

Antioxidant enzymes are a class of active subtances widely found in animals, plants and microorganisms,including peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX). Its main function is to catalyze the oxidation of hydrogen peroxide and other peroxides to various organic and inorganic substances, and to convert peroxides into less toxic or harmless substances by redox reaction (Liet al.,2013). POD is one of the key enzymes of plant enzymatic defense system under adverse conditions,which coordinates with CAT to remove excess free radicals. The high concentration of H2O2in the tissue requires CAT to remove and the low concentration is mainly eliminated by POD in the oxidation of the corresponding matrix, so that the free radicals in the body are maintained at a normal dynamic level to improve the plant's stress resistance. In general,peroxidase activity is positively correlated with resistance and high resistance to peroxidase activity is also high. CAT is also one of the protective enzymes to eliminate oxygen free radicals in cells, which catalyzes the decomposition of H2O2into H2O and O2. CAT mainly exists in cell peroxisomes and is responsible for the clearance of H2O2produced in peroxidation.CAT can also decompose and remove H2O2produced in other parts and diffuse into peroxisomes, which plays an important role in plant respiratory metabolism(Jinet al., 2009). APX is a hemoglobin composed of a peptide chain and a porphyrin, and apoprotein molecule binds to haem to form a holoenzyme. APX catalyzed reaction is 2AsA+H2O2→2MDA+2H2O. In some organelles, the ascorbate glutathione cycle (AsAGSH) effectively removes H2O2produced by cell metabolism in the absence of catalase. It is generally believed that APX is the main enzyme that cleans H2O2in chloroplasts.

As early as 1997, Houet al.(1997) found that lanthanum and cerium treated wheat reduced the activities of peroxidase isoenzymes, especially the activity of peroxidase, which was involved in auxin catabolism and the activity of peroxidase was slow,thus promoted plant growth. Studies showed that 10 mg · L-1of Ce can alleviate the toxicity of Cd on maize seeds, increase seed germination rate, enhanceα-amylase, SOD, POD and CAT activities and decrease MDA content and cytoplasmic membrane permeability(Liet al., 2013).

Three soybean varieties of different quality types in Northeast China were used as experimental materials and different concentrations of LaCl3, CeCl3and LaCl3+CeCl3solutions were sprayed on the leaves at the seedling stage and the early fl owering stage, respectively. The activities of POD, CAT and APX in the leaves of plants were determined and the effects of rare earth on the physiological activities of soybean were investigated. Through this experiment to study the effect of rare earth on the antioxidant enzyme activities of different quality soybeans in Northeast China, it would provide a theoretical basis for guiding scienti fic and rational application of rare earth to soybeans.

Materials and Methods

Materials

Soybeans were chosen from Soybean Research Institute of Northeast Agricultural University, they were Dongnong 42 (high protein), Dongnong 47(high fat) and Dongnong 52 (mixed-use), which were planted in garden pots with the height of 30 cm,diameter of 35 cm at the top and 25 cm at the bottom.Each pot contained 15 kg of black soil and eight full and uniformly-sized seeds were sown in each pot. Four seedlings were kept, when the seedlings grew to 5-8 cm. The potted plants were grown using conventional methods and the black soil was derived from the Horticultural Experiment Station of Northeast Agricultural University. The chemical properties of the soil used for testing provided organic matter 31.3 g · kg-1, pH (H2O) 7.62, available nitrogen 147 mg · kg-1, available phosphorus 74 mg · kg-1,available potassium 202 mg · kg-1and the total nitrogen 3.58 mg · kg-1.

Treatments

The periods of spraying lanthanum and cerium solutions were the soybean seedling stage (June 17th,2017) and the early fl owering stage (July 1th, 2017).Spraying concentrations of different kinds of rare earth solutions were selected according to the preliminary test. The following concentrations were set for the experiment: the first group of 30, 60 and 90 mg · L-1CeCl3solutions (represented by Ce3, Ce6 and Ce9,respectively), the second group with concentrations of 120, 150, 180 mg · L-1LaCl3solutions (represented by La12, La15 and La18, respectively) and the groups of 30, 40, 60 mg · L-1, CeCl3and LaCl3, according to a 1 : 1 ratio volume mixing solution (represented by L+C3, L+C4 and L+C6, respectively), and then these solutions were sprayed on all the soybean leaves. The control group (CK) was sprayed with the same dose of distilled water. Each sample had three replications.

The functional leaves of soybeans were obtained on the 7th day after spraying lanthanum and cerium in the early flowering stage. The picked leaves were wiped clean and immediately frozen in liquid nitrogen, then were transferred to a 80℃ refrigerator for determination of antioxidant enzymes.

Measurement indicators and methods

The activity of peroxidase (POD) was determined by guaiacol (GA) colorimetry (Shanghai Society of Plant Physiology, 1999), it was based on the detection of the absorbance of product at 470 nm in a reaction system of the guaiacol. The activity of catalase(CAT) was determined by UV spectrophotometry at 470 nm wavelength (Chen, 2000). The ascorbate peroxidase activity (APX) was determined by UV spectrophotometry at 290 nm wavelength (Zou, 1997).

Data processing and analysis

Statistical analyses were carried out using Microsoftexcel software 2007. Statistical analyses were performed using one-way analysis of variance (ANOVA)and the group means were compared by Duncan test using SPSS software 19.0.

Results

Effect of lanthanum and cerium on POD activity of soybean leaves

As shown in Fig. 1, with the increase of LaCl3concentration, POD activity of Dongnong 42 leaves decreased gradually and was lower than that of CK. In the treatment range of 120-180 mg · L-1, POD activity of soybean leaves decreased significantly by 28.55%,30.24% and 59.59% compared with that of CK. With the increase of CeCl3concentration, the activity of POD increased, and Ce9 was the highest, which was 6.72%higher than that of CK (p＞0.05). With the increase of LaCl3+CeCl3concentration, POD activity increased gradually and L+C6 was up to 328.33 U · g-1FW · min-1,with a significant increase of 11.36% (p＜0.05). In general, the concentration of lanthanum and cerium set in this experiment was not good for improving POD activity of Dongnong 42 leaves, but the effects of L+C6 and Ce9 were better.

Fig. 1 Effect of lanthanum and cerium on POD activity in leaves of soybean Dongnong 42

As shown in Fig. 2, POD activity of Dongnong 47 leaves decreased with the increase of lanthanum concentration and was higher than that of CK, only the treatment of La12 was higher than CK and significantly increased by 13.94% (p＜0.05). POD activity increased firstly and then decreased with the increase of CeCl3concentration, at Ce6 treatment was 19.91%(p＜0.05) higher than that of CK. LaCl3+CeCl3group showed a trend which first increased and then decreased with increasing concentration. The activity of POD was the highest under L+C4 treatment, which was 15.80% (p＜0.05) higher than that of CK. The results showed that both lanthanum and cerium promoted POD activity of Dongnong 47 leaves, but its activity was seriously inhibited from La treatment group due to the increased concentration.

Fig. 2 Effect of lanthanum and cerium on POD activity in leaves of soybean Dongnong 47

As shown in Fig. 3, with the increase of LaCl3concentration, POD activity of Dongnong 52 increased first and then decreased. Under La15 treatment, POD activity was higher than that of CK and significantly increased by 22.77% (p＜0.05). Under CeCl3treatment, POD activity increased with the increase of concentration and reached the peak under Ce9 treatment,which was significantly higher than that of CK by 27.16% (p＜0.05). LaCl3+CeCl3group showed an increased trend with increasing concentration and POD activity was the highest under L+C6 treatment, which was significantly increased by 33.49% compared with that of CK. Overall, the mixed lanthanum and cerium was better than the single rare earth to improve POD activity of Dongnong 52.

Fig. 3 Effect of lanthanum and cerium on POD activity in leaves of soybean Dongnong 52

Effect of lanthanum and cerium on CAT activity of soybean leaves

As shown in Fig. 4, with the increase of LaCl3concentration, CAT activity gradually decreased and was lower than that of CK. It was because La concentration was too high that CAT could no longer clear the excessive H2O2produced by the stress in the plants.The enzyme activity increased with the increase of CeCl3concentration and reached the peak value under Ce9 treatment, which was 18.50% (p＞0.05) higher than that of CK. With the increase of LaCl3+CeCl3concentration, the activity of CAT decreased first and then increased, and only increased 13.89% compared with that of CK under the treatment of L+C6 (p＞0.05).The results showed that the application of LaCl3in Dongnong 42 significantly reduced the activity of CAT in the treatment range of 120-180 mg · L-1,indicating that high concentration of rare earth La would destroy the antioxidant system, which was not conducive to plants to resist and repair the damage caused by external environment.

As shown in Fig. 5, CAT activity of Dongnong 47 leaves gradually decreased and was lower than that of CK with the increase of LaCl3concentration and reached the maximum under La12 treatment and increased 12.73% (p＞0.05) compared with that of CK.CAT activity of CeCl3group gradually decreased,which reached the highest at Ce6 and increased 8.48%(p＞0.05) compared with that of CK. The activity of CAT increased firstly and then decreased after LaCl3+CeCl3treatment and reached the peak at LC4,the activity of CAT was the best at this concentration and the activity of CAT was significantly increased by 51.52% (p＜0.05). In general, LaCl3+CeCl3solution significantly improved CAT activity of Dongnong 47 leaves, which was better than LaCl3and CeCl3.

Fig. 4 Effect of lanthanum and cerium on CAT activity in leaves of soybean Dongnong 42

Fig. 5 Effect of lanthanum and cerium on CAT activity in leaves soybean Dongnong 47

As shown in Fig. 6, with the increase of LaCl3concentration, CAT activity increased first and then decreased, reached the maximum under La15 treatment and was 7.7% higher than that of CK (p＞0.05). In the CeCl3group, the enzyme activity increased with the increase of concentration and CAT activity reached the highest at the treatment concentration of Ce9, which was 23.1% higher than that of CK (p＞0.05). Under the condition of LaCl3+CeCl3treatment, the peak value at L+C6 was 19.23% higher than that of CK (p＞0.05).The best effect of increasing the activity of CAT was 15.5 U · g-1FW · min-1at this concentration (p＞0.05).Overall, the activity of CAT of Dongnong 52 was not significantly improved under the treatment of this experiment.

Fig. 6 Effect of lanthanum and cerium on CAT activity in leaves soybean Dongnong 52

Effect of lanthanum and cerium on APX activity in soybean leaves

As shown in Fig. 7, APX activity gradually decreased with the increase of LaCl3concentration and reached the highest level at La12, which was 3.68% higher than that of CK (p＜0.05), other concentrations were lower than those of CK. In CeCl3treatment group,the activity of APX increased with the increasing concentration, reached the highest at Ce9 and significantly increased by 6.36% compared with that of CK(p＜0.05). The activity of APX increased significantly during LaCl3+CeCl3treatment and was significantly higher than that of CK. The highest value reached 1.41 U · min-1· mg-1at L+C6 and increased 50.00%than that of CK (p＜0.05). Thus, the low concentration of LaCl3could promote APX activity of Dongnong 42 leaves, while the high concentration produced inhibition, indicating that it had the phenomenon of"low promotion and high inhibition".

As shown in Fig. 8, with the increase of LaCl3concentration, APX activity of Dongnong 47 leaves gradually decreased and reached the highest at La12, up 22.64% from that of CK (p＞0.05). Under the increase of CeCl3concentration, APX activity increased first and then decreased and reached the highest at Ce6 increased by 11.35% compared with that of CK (p＞0.05). Under LaCl3+CeCl3treatment,APX activity increased first and then decreased and reached a peak increased by 20.75% at L+C4(p＞0.05). It could be seen that spraying rare earth La and Ce at the seedling stage and the early flowering stage increased APX activity of Dongnong 47 leaves,although the difference was not significant with the control, it was consistent with the phenomenon of "low promotion and high inhibition".

Fig. 7 Effect of lanthanum and cerium on APX activity in leaves of soybean Dongnong 42

Fig. 8 Effect of lanthanum and cerium on APX activity in leaves of soybean Dongnong 47

As shown in Fig. 9, APX activity of the leaves of Dongnong 52 increased first and then decreased under the treatment of LaCl3concentration and reached the highest in La15, increased by 11.76% compared with that of CK (p＞0.05). In CeCl3treatment group, APX activity was the highest in Ce6 and 13.73% higher than that of CK (p＞0.05). The activity of APX increased with the increase of concentration in LaCl3+CeCl3treatment and both higher than that of CK. Under L+C6 treatment, it increased by 23.53% compared with that of CK and reached the maximum value. In general, APX activity of Dongnong 52 leaves was not significantly increased by spraying rare earth, the mixed rare earth was better.

Fig. 9 Effect of lanthanum and cerium on APX activity in leaves of soybean Dongnong 52

Discussion

Rare earth promoted the growth and development of plants (Zhaoet al., 2014) mainly in two aspects: on the one hand, lanthanum and cerium could improve the photosynthetic organs and activities of plants, increase the activity of enzymes in plants, and thus improve the photosynthesis ability of plants (He and Xue, 2005);on the other hand, it could promote seed germination and increase seed emergence rate. The rare earth in promoting seed germination had been successfully applied to wheat and other field crops. For example,the germination rate of wheat could be increased from 8% to 19%, and the germination rate of fl ax could be increased from 7% to 12% (Liu, 2004). The experimental study showed that the appropriate concentration of lanthanum and cerium could increase the antioxidant enzyme activity of soybean leaves and reduce the damage caused by excessive concentration of free radicals and reactive oxygen species. The results of this study were similar to those of others,Wanget al.(2013) found that CeCl3could enhance the ability of pea cells to scavenge reactive oxygen species and the low concentration of rare earth could protect the antioxidant enzyme system of pea seedlings under heavy metal copper stress.

Many experiments showed that rare earth ions could interact with a variety of biological proteins and enzymes and affect their physiological activities (Wanget al., 2013). Other studies also showed that lanthanum could increase the activities of antioxidant enzymes SOD and CAT not only in the normal growth of wheat seedlings, but also increase the activities of SOD and CAT and reduce the content of MDA in wheat seedlings at the early stage of mercury stress (Panget al., 2002), which was similar to the results of this study, according to the analysis of Dongnong 42, with the increase of LaCl3concentration, the activities of CAT, POD and APX decreased and were lower than those of CK, which was a serious inhibitory effect.However, APX activity was higher than that of CK at La12 concentration, which might be caused by plant stress response. In CeCl3and LaCl3+CeCl3treatment groups, the activity trends of the three enzymes were consistent, which increased with increasing concentration, peaked at Ce9 and L+C6 concentrations and the enzyme activity was increased to the maximum extent. Comprehensive analysis, for the high protein variety Dongnong 42, the mixed lanthanum and cerium was better than the single rare earth lanthanum and single rare earth cerium. The trends of CAT, POD and APX in the leaves of Dongnong 47 sprayed with LaCl3group were the same and decreased with the increase of concentration. The activities of the three enzymes were higher than those of CK at La12, which could increase the activities of enzymes at this concentration,but decreased them at higher concentration. In CeCl3and LaCl3+CeCl3groups, the three enzyme activities showed the same trend, which increased first and then decreased and the phenomenon of "low promotion and high inhibition" was obvious. Other studies also found that high concentration of lanthanum and cerium aggravated the damage of Cu to the antioxidant enzyme system of pea seedlings and showed the toxic effect of synergistic stress with Cu ion (Wanget al.,2013). Studies showed that when La3+was sprayed to rapeseed with a concentration higher than 30 mg · L-1, the chlorophyll content decreased and led to the leaves chlorosis, which manifested that the activity of nitrate reductase decreased, the root activity decreased and finally the biomass of rapeseed decreased (Zenget al.,2001). According to comprehensive analysis, for the high-fat variety Dongnong 47, mixed rare earth had better enzyme activity. For Dongnong 52, with the increase of LaCl3concentration, the activities of the three enzymes increased first and then decreased,while in CeCl3and LaCl3+CeCl3treatment groups, the three enzymes showed the same change trend and all increased with the increase of the concentration. The data showed that the mixed rare earth was better for improving enzyme activity of Dongnong 52.

Therefore, when using rare earth as elements to regulate crop growth, it was necessary to pay attention to the use of effective doses, which might result in losses in crop production. Excessive LaCl3caused dysfunction of the antioxidant defense system in plants,leading to enhanced membrane lipid peroxidation in soybean leaves, thereby inhibiting the activities of protective enzymes and reducing antioxidant content(Renet al., 2017). It indicated that the use of rare earth elements should pay attention to the spraying time and other conditions and factors and paying attention to the effective dose. Only the better cooperation could be applied to maximize the beneficial effects of rare earth. This aspect of the research was not covered in this paper and needed further studies. In addition,whether the ability of Ce to promote the degradation of superoxide free radical was definitely higher than that of La, more experimental conditions should be set to con firm it and the mechanism of reducing stress should be further studied.

Conclusions

The suitable concentration of rare earth could increase the antioxidant enzyme activity of soybean leaves.Different kinds and concentrations of rare earth had different effects on soybeans, and the same concentration and kind of rare earth had different effects on different quality soybeans. Spraying lanthanum and cerium at the seedling stage and the early flowering stage, the mixed rare earth increased the activities of the three enzymes, which was better than the single rare earth.

The optimum rare earth for Dongong 42 was the mixed solution concentration of lanthanum and cerium of 60 mg · L-1, compared with CK, the activities of peroxidase, catalase and ascorbate peroxidase increased by 11.36%, 13.89% and 50.00%, respectively.

The optimum rare earth for Dongong 47 was the mixed solution concentration of lanthanum and cerium of 40 mg · L-1, compared with CK, the activities of peroxidase, catalase and ascorbate peroxidase increased by 15.80%, 51.52% and 20.75%, respectively.

The optimum rare earth for Dongong 52 was the mixed solution concentration of lanthanum and cerium of 60 mg · L-1, compared with CK, the activities of peroxidase, catalase and ascorbate peroxidase increased by 33.49%, 19.23% and 23.53%, respectively.