Effects of Exogenous 5-Aminolevulinic Acid and 24-Epibrassinolide on Cd Accumulation in Rice from Cd-Contaminated Soil

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S cienceD irect R ice S cience, 2018, 25(6): 320−329

Effects of Exogenous 5-Aminolevulinic Acid and 24-Epibrassinolide on Cd Accumulation in Rice from Cd-Contaminated Soil WANG Feijuan1, #, ZHANG Yiting1, #, GUO Qinxin1, TAN Haifeng1, HAN Jiahui1, LIN Haoran1, WEI Hewen2, XU Guangwei2, ZHU Cheng1 (1Key Laboratory of Marine Food Quality and Hazard Controlling Technology of Zhejiang Province, College of Life Sciences, China Jiliang University, Hangzhou 310018, China; 2Jinhua Inspection and Testing Institute of Food and Drug Control, Jinhua 321000, China; # These authors contribute equally to this study)

Abstract: H igh grain-C d-acc umulating rice variety Y ongyou 9 was planted in C d-contaminated farmland in T aizhou C ity, Z hejiang P rovince, C hina to study the effects of 5-aminolevulinic acid (A L A ) and 24-epibrassinolide (E B R ) on C d accumulation in brown rice. R esults showed that the exogenous A L A and E B R had no significant effects on agronomic traits, soil pH and total C d content in soil, but had some effects on the available C d content in soil, and significantly influenced the C d accumulation in the different parts of rice. R esults also showed that 100 mg/L exogenous A L A significantly reduced the C d accumulation in brown rice to blow the food safety standard (0.2 mg/kg), and also significantly reduced the C d contents in the roots and culm of rice. H owever, 200 mg/L exogenous A L A treatment increased the C d content in brown rice remarkably. In addition, 0.15 mg/L E B R treatment increased C d accumulation in roots, culm, leaves and brown rice notably, whereas 0.30 mg/L exogenous E B R treatment reduced the C d accumulation in brown rice properly, but it was not significant. T herefore, proper concentration of A L A can effectively reduce the C d accumulation in brown rice, which can be used as an effective technical method for the safe production of rice in C d polluted farmland. Key words: rice; C d-contaminated soil; 5-aminolevulinic acid; 24-epibrassinolide; C d accumulation

Rice (Oryza sativa L.) is one of the most important cereal crops in the world. Cd pollution in rice is highly concealed and dangerous (Sharma et al, 2007; Wang et al, 2014), posing great risks and harms to the safe production of rice grain and peopleʼs health (Liu et al, 2007; Grant and Sheppard, 2008; Six, 2010). In recent years, excessive Cd content in rice and its processing of agricultural products are often detected. Researchers found that nearly 10% of Chinese commercial rice grains have high Cd accumulations, which exceeds the food safety standard (0.2 mg/kg). Cd-polluted farmland in China exceeds 1.3 × 105 km2, and Cd-contaminated rice grain reaches 5.0 × 104 t (Wang, 2002). Liu et al

(2016) also showed that the heavy metal Cd pollution in rice grains in some parts of the Yangtze River basin in China is relatively serious. Under Cd stress, rice exhibits complex changes at the physiological, biochemical and molecular levels. The main symptoms are as follows: slowly grow, chlorophyll loses, leaves and roots necrosis, stomata function changes (Liu C F et al, 2011; Xue et al, 2013), the structural of partial organs and ultrastructural changes (Shi and Cai, 2008), leaf transpiration and photosynthesis inhibition (Mobin and Khan, 2007; Shi and Cai, 2008; Liu C F et al, 2011). At the same time, Cd is easily absorbed and accumulated in different parts of rice (Fang et al, 2014;

Received: 1 April 2018; Accepted: 26 July 2018 Corresponding author: WANG Feijuan ([email protected]) Copyright V Copyright © © 2018, 2018, China China National National Rice Rice Research Research Institute. Institute. Hosting Hosting by by Elsevier Elsevier B B.V. This This is is an an open open access access article article under under the the CC CC BY-NC-ND BY-NC-ND license license (http://creativecommons.org/licenses/by-nc-nd/4.0/) (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer Peer review review under under responsibility responsibility of of China China National National Rice Rice Research Research Institute Institute http://dx.doi.org/ http://dx.doi.org/10.1016/j.rsci.2018.10.002

WANG Feijuan, et al. Effects of Exogenous ALA and EBR Treatments on Cd Stressed Rice

Kosolsaksakul et al, 2014), which in turn can cause serious harm to human and animal health through the food chain (Hayat Q et al, 2010; Recatalá et al, 2010). Therefore, limiting Cd content in rice grains is an urgent problem to be solved. The remediation of heavy-metal-contaminated soils with the method of applying improvers is a commonly used improvement measure (Krystofova et al, 2012). A large number of reports showed that the application of different modifiers can effectively passivate the heavy metals in soil (Six, 2010; White et al, 2012; Ali et al, 2015). The accumulation of Cd in rice grains can be effectively controlled by regulation of fertilizer (Shao et al, 2010), lime control of soil pH (Tavakkoli et al, 2011) and regulation of soil redox state (Gayomba et al, 2016). Studies have shown that soil pH has some effects on the accumulations of nutrient elements and heavy metals (Hong et al, 2014; Zhang et al, 2014). These pathways could control the accumulation of Cd in rice grains, however, have many uncertain effects on rice. At the same time, different exogenous substances applied to reduce the Cd content in rice grains have also been gradually carried out. Applying exogenous organic acids (Xin et al, 2015) and ethylenediaminetetraacetic acid (EDTA) (Huang et al, 2018) to Cd-contaminated soil reduces Cd content in brown rice, indicating that organic acids and EDTA inhibit the transfer and accumulation of Cd to rice grains (Xin et al, 2015; Huang et al, 2018). The synthesis of 5-aminolevulinic acid (ALA) affects the rate of chlorophyll synthesis in plants, which is synthesized from glutamate in a reaction involving a glutamyl-tRNA intermediate (Richter et al, 2010; Nahar and Shimasaki, 2014). Foliar applications of exogenous ALA can improve the plant growth, chlorophyll biosynthesis and the photosynthesis, then increase the crop yield (Zhang C P et al, 2013). As a potential plant growth regulator, a certain concentration of ALA is effective in counteracting the harmful effects of various abiotic stresses in plants (Akram et al, 2013). When the plant is subjected to biotic and abiotic stresses, 24-epibrassinolide (EBR) has a strong regulatory effect as a bioregulator (Sharma et al, 2015). Foliar applications of exogenous EBR influence cell permeability, and heavy metal absorption by acting on the electrical properties and enzyme activities of the membrane (Rady, 2011; Ramakrishna and Rao, 2012; Fariduddin et al, 2013). In addition, due to the higher activity of ATPase, the toxicity of heavy metals reduced is related to the production of soluble proteins

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and nucleic acids (Choudhary et al, 2011; Ramakrishna and Rao, 2012). Shahzad et al (2018) reviewed the role of EBR in pesticide metabolism and detoxification. EBR can detoxify the toxic effects induced by pesticides and mitigate heavy metals by enhancing the antioxidant defense system and reducing its residual amount in plants. If EBR was administered at an appropriate concentration and at an appropriate stage under stress conditions, it could exert an immunomodulatory effect in plants. In this study, foliar applications of exogenous ALA and EBR to rice seedlings grown on Cd-contaminated farmland were conducted to investigate the effects of ALA and EBR on the Cd accumulation in different parts of rice, trying to provide technical support for the safe production of rice in medium and mild Cd-contaminated farmland.

MATERIALS AND METHODS Materials The tested rice variety was Yongyou 9, which was pre-screened as a high grain-Cd-accumulating rice in the early research (Shi et al, 2016). Experimental treatments One control (CK, distilled water) and four treatments (100 and 200 mg/L ALA, 0.15 and 0.30 mg/L EBR) were used. The different substances were separately sprayed on the rice leaves at two weeks after transplanting, tillering, heading and flowering stages, respectively, until the whole leaves were covered by the solutions. A total of 12 m2 and 3 L related solution were used for each treatment with three repeats. Conventional water and fertilizer management were carried out during the field plot experiment in the Cd-contaminated soil of Taizhou in Zhejiang Province, China (from April to November in 2016). All water, fertilizer and pesticide managements were conducted according to the normal farmer practice. A total of 150 kg/hm2 nitrogen were applied at the transplanting, tillering and heading stages with the ratio of 2:2:1. Nitrogen (N), phosphorus (P2O5) and potassium (K2O) fertilizers were applied at a ratio of 1:0.3:0.6. Phosphate fertilizer was applied once at the transplanting stage, whereas potassium fertilizer was applied two times at the transplanting and heading stages. Rice was planted with 0.5 m interval among different treatments, with spacing of 0.3 m ×0.2 m.

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The land between each treatment area was covered with a plastic film to prevent cross-linking in water and fertilizer. The soil pH, total Cd, organic matter, alkali nitrogen, available phosphorus and available potassium were 5.56, 1.27 mg/kg, 59.3 g/kg, 271 mg/kg, 21.6 mg/kg and 60.0 mg/kg, respectively. Investigation on agronomic traits of rice Rice samples were taken at the ripening stage, five mature rice plants of each treatment were selected for agronomic traits analysis, including plant height, number of spikelets per plant, number of grains per panicle, 1000-grain weight and seed-setting rate. In order to remove Cd ions adsorbed on the root surface, the roots of the harvested mature rice plants were all treated with 20 mmol/L ethylenediaminetetraacetic acid disodium (Na2-EDTA) solution for about 30 min, then rinsed repeatedly with distilled water for several times. The mature rice plants were divided into four parts: root, culm, leaf and spikelet. The root, culm and leaf samples were washed with tap water and distilled water, oven dried at 105 oC for 30 min and baked at 70 oC to constant weight, then crushed into powder. The spikelets of rice plants were divided into husk and brown rice, and then crushed to powder, respectively. Determination of soil pH The sampled soil was naturally air-dried and removed through a 2.5 mm nylon screen to remove grit and plant debris, then passed through a 100-mesh screen and stored. Potentiometric method was used to determine soil pH according to Hu and Cao (2007) with some modifications. The pretreated soil sample (10.0 g) and 25 mL CaCl2 (0.01 mol/L) solution were mixed well with a glass rod for 1–2 min, standing for 30 min, and then measured with a pH meter. Determination of available Cd in soil Available Cd was extracted with diethylenetriaminepentaacetic acid (DTPA) solution [DTPA (0.005 mol/L), CaCl2 (0.01 mol/L) and triethanolamine (0.1 mol/L), pH 7.3]. Soil sample (2.50 g) and 25 mL DTPA solution were mixed and shocked at 25 ºC at the speed of 180 r/min for 1 h. After filtering, 1 mL filtrate was diluted to 10 mL with 1% HNO3 solution. The available Cd was determined by an atomic absorption spectrometer (AA7000, SHIMADZU, Kyoto, Japan). Determination of total Cd in soil Soil samples (0.2 g) were digested in an acid mixture

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of HNO3-HF (4:2), using a microwave digester (CEM-MARS, Boston, USA) with a procedure showed in Table 1. Digestion tubes were then held on an electric heating plate until the solution became clear light yellow color and less than 1 mL. The digestion solutions were subsequently moved to 25 mL volumetric flasks. Cd content in soil was determined with an atomic absorption spectrometer (AA7000, SHIMADZU, Kyoto, Japan). Determinations of Cd content in different parts of rice plants and other mineral elements in brown rice Different parts of rice samples (0.3 g) were evaluated and placed into digestion vessels. The vessels were stored in a ventilation cabinet for 2–3 h after 6 mL high purity HNO3 was added, and then digested in a microwave digester (CEM-MARS, Boston, USA). The procedure was shown in Table 1. After digestion, all the mixtures were driven acid to 1 mL at elevated temperature. The solutions were then diluted into 25 mL volumetric flasks with ultrapure water, and the clarified samples thus obtained were kept in a refrigerator at 4 ºC for further analysis. Cd content was determined by an atomic absorption spectrometer (AA7000, SHIMADZU, Kyoto, Japan). Other mineral elements (Fe, Ni, Mn, Se, Zn, Cu and Pb) were analyzed by using inductively coupled plasma mass spectrometry (ICP-MS) according to Wang and Li (2010). Statistical analysis Data were analyzed using the Excel 2007 and SPSS (Product and Service Solutions Statistical, 18.0), and at least three replicates of the samples were prepared.

RESULTS Effects of ALA and EBR on agronomic traits of rice It was found that only exogenous EBR treatment could reduce the seed-setting rate, but had no significant effect on other agronomic traits (plant height, number of spikelets per plant, number of Table 1. Microwave digestion procedure of samples. Power (W) 1600

Heating up time (min) 5

Temperature (ºC) 120

Continuous time (min) 5

1600

5

170

15

1600

5

190

10

WANG Feijuan, et al. Effects of Exogenous ALA and EBR Treatments on Cd Stressed Rice

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Table 2. Effects of different exogenous substance treatments on agronomic traits of rice in Cd contaminated paddy fields. Treatment

Plant height (cm)

No. of spikelets per plant

No. of grains per plant

1000-grain weight (g)

Seed-setting rate (%)

CK 127.75 ± 1.06 a 15.5 ± 0.7 ab 3 400.7 ± 207.8 ab 26.32 ± 0.55 a 100 mg/L ALA 124.83 ± 2.36 a 18.2 ± 0.3 a 3 688.8 ± 164.0 a 26.10 ± 0.47 a 200 mg/L ALA 125.67 ± 0.76 a 15.8 ± 0.6 ab 3 195.0 ± 85.2 ab 26.16 ± 0.51 a 0.15 mg/L EBR 127.00 ± 2.65 a 15.5 ± 3.1 ab 3 547.0 ± 381.7 ab 26.02 ± 0.57 a 0.30 mg/L EBR 127.00 ± 3.18 a 14.8 ± 1.4 b 3 216.2 ± 478.0 ab 26.27 ± 0.27 a ALA, 5-aminolevulinic acid; EBR, 24-epibrassinolide. Values are mean ± SD (n = 5). The same lowercase letters in the same column indicate no significant difference at P < 0.05. Table

3. Effects of 5-aminolevulinic acid (ALA) and 24-epibrassinolide (EBR) treatments on soil pH values.

Treatment

soil and change the Cd content, which could be effectively absorbed by other organisms, and thereby change the Cd uptake progress in rice.

pH value

CK 5.56 ± 0.06 a 100 mg/L ALA 5.60 ± 0.04 a 200 mg/L ALA 5.55 ± 0.07 a 0.15 mg/L EBR 5.56 ± 0.11 a 0.30 mg/L EBR 5.64 ± 0.03 a Values are mean ± SD (n = 5). The same lowercase letters in the same column indicate no significant difference at P < 0.05.

Effects of ALA on Cd accumulation in different parts of rice plants From Fig. 2, we found that the Cd content in CK was about 0.25 mg/kg. Under 100 mg/L ALA treatment, the Cd content in brown rice was about 0.18 mg/kg, 38% lower than the control, which reached the edible standard below the food safety standard in China. However, the Cd accumulation in brown rice was significantly increased under 200 mg/L ALA treatment. Fig. 2 showed the effects of ALA treatments on Cd concentrations in roots, culm and leaves of rice. Exogenous ALA (100 mg/L) treatment could effectively reduce the Cd concentrations in roots and culm of rice by 27.3% and 25.7%, respectively. However, 200 mg/L ALA had no significant effect on the Cd concentrations in roots, culm and leaves of rice, but increased the Cd concentration in the brown rice (Fig. 2).

grains per plant and 1000-grain weight). Exogenous ALA treatment had no significant impact on any of these indicators (Table 2). Effects of ALA and EBR on soil physicochemical properties ALA and EBR had little effects on the pH value (Table 3) and total Cd content in the soil (Fig. 1). Compared with CK, the available Cd content in the soil decreased significantly under 100 mg/L ALA treatment (reduced about 18%) (Fig. 1-A). However, after treatment with the higher concentration (200 mg/L) ALA, the effective Cd content in the soil increased (Fig. 1-A). At the same time, the Cd concentration in soil was also affected by EBR treatment. The available Cd content in the soil was increased by 35.7% under EBR treatment compared with CK (Fig. 1-B). In general, the exogenous ALA and EBR treatments could affect the form of Cd in the A Cd content (mg/kg)

Effects of EBR on Cd accumulation in different parts of rice plants EBR treatments might affect the migration and distribution of Cd in rice, and Cd accumulations in mature rice plants were showed: root > culm > brown rice > leaf, indicating that root is the main organ for B

a

a

a b

a

a

a

95.46 ± 0.02 a 93.28 ± 0.01 ab 93.83 ± 0.01 ab 91.37 ± 0.06 b 91.46 ± 0.03 b

b

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a

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a

Fig. 1. Effects of 5-aminolevulinic acid (ALA, A) and 24-epibrassinolide (EBR, B) on Cd content in soil. Values are mean ± SD (n = 9). Different lowercase letters above the different columns indicate significant difference at P < 0.05.

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Fig. 2. Effects of 5-aminolevulinic acid (ALA) on Cd content in different parts of rice plants. Values are mean ± SD (n = 9). Different lowercase letters above the different columns indicate significant difference at P < 0.05.

Fig. 3. Effects of 24-epibrassinolide (EBR) on Cd content in different parts of rice plant. Values are mean ± SD (n = 9). The same lowercase letters above the different columns indicate no significant difference at P < 0.05.

Cd uptake in rice (Fig. 3). After treatment with 0.15 mg/L of exogenous EBR, the Cd content in brown rice increased obviously, at about 0.35 mg/kg, 41.67% more than CK. However, 0.30 mg/L exogenous EBR reduced the Cd accumulation in brown rice to a degree, but it was not significant (Fig. 3). The Cd contents in brown rice under EBR treatment were all higher than 0.2 mg/kg (the edible standard below the food safety standard in China). The Cd contents in CK, 0.15 mg/L and 0.30 mg/L EBR treatments exceeded the limit of 0.2 mg/kg by 20%, 70% and 15%, respectively. EBR treatment (0.30 mg/L) could reduce the Cd concentration in brown rice properly, but it was not significant. EBR treatment could also affect the Cd accumulation in different parts of rice plants. Compared with CK, Cd accumulation in roots, culm and leaves increased after treated with different concentrations of EBR (Fig. 3). After treatment with 0.15 mg/L EBR, Cd contents in roots, culm and leaves increased significantly, while 0.30 mg/L EBR treatment showed no significant effect on the Cd concentration in roots and leaves, but it obviously increased the Cd concentration in culm.

significant up-regulation nor significant reduction (Table 4). The results showed that the treatment of exogenous ALA only had a significant effect on the accumulation of Cd in rice, but had no significant effect on other metal elements.

Effects of ALA on accumulations of other mineral elements in brown rice The experimental results showed that after foliar application with ALA, there was no significant change in other metal elements in brown rice, neither

DISCUSSION Cd inhibits rice growth, such as plant dwarfism, and results in biomass loss (Dubey et al, 2014; Li et al, 2016). ALA is a potential plant growth regulator, and application with the proper concentration of ALA is effective against the harmful effects of various abiotic stresses in plants (Akram and Ashraf, 2013). Zhang et al (2008) showed that application of ALA significantly improves the plant growth under herbicide stress in Brassica napus. Moreover, exogenous ALA treatment significantly increases the fresh and dry weights of roots and culm to enhance plant growth under salinity stress conditions (Naeem et al, 2010, 2012). Brassinosteroids (BRs) are very helpful for plant growth and development in root inhibition, ethylene biosynthesis, senescence, photosynthesis and enzyme activation (Bajguz and Hayat, 2009; Hayat et al, 2011). EBR is an active brassinosteroid, which has the same role as BRs (Zhang Y P et al, 2013). Therefore, in this study, the agronomic traits of mature rice were investigated. The experiment results indicated that only the EBR treatment could affect the seed-setting

Table 4. Effects of 5-aminolevulinic acid (ALA) treatments on other mineral elements in brown rice. Treatment

Fe (mg/kg)

Ni (μg/kg)

Mn (mg/kg)

Se (μg/kg)

Zn (mg/kg)

Cu (mg/kg)

CK 21.80 ± 1.54 a 125.64 ± 9.09 a 36.65 ± 3.49 a 23.87 ± 0.43 a 23.07 ± 1.08 a 9.22 ± 0.62 a 100 mg/L ALA 21.22 ± 3.35 a 127.67 ± 8.51 a 36.37 ± 1.17 a 23.47 ± 1.68 a 22.78 ± 1.03 a 9.12 ± 1.02 a 200 mg/L ALA 21.12 ± 2.76 a 125.84 ± 15.44 a 36.80 ± 2.49 a 23.90 ± 0.66 a 23.60 ± 0.82 a 9.15 ± 0.64 a Values are mean ± SD (n = 6). Different lowercase letters in the same column indicate significant difference at P < 0.05.

Pb (μg/kg) 83.07 ± 11.90 a 81.90 ± 6.91 a 81.11 ± 7.98 a

WANG Feijuan, et al. Effects of Exogenous ALA and EBR Treatments on Cd Stressed Rice

rate of rice. Treatment with different concentrations of ALA and EBR had no significant effects on the other agronomic traits of rice. The transport mechanism of Cd in rice is now pretty clear. First, Cd is uptaken by plant roots through the corresponding transport proteins. As Cd is a nonessential ion, there are no specific Cd transporters. However, many of transporters for divalent transition metals (Mn, Fe and Zn) can uptake Cd (MendozaCozatl et al, 2011; Uraguchi and Fujiwara, 2012). Second, Cd transport was influenced by Cd chelation and compartmentation in vacuoles. Because of the high toxicity of Cd, plants have evolved adaptive mechanisms through detoxification of accumulated Cd through different pathways (Lanquar et al, 2005; Marentes and Rauser, 2007). Third, Cd is transported from root to shoot with long distance through the xylem (Ueno et al, 2009; Song et al, 2011). Finally, Cd transport from shoots to grains. In rice, almost 100% of the Cd deposition into grains is mediated by phloem. According to Kashiwagi et al (2009), the accumulation of Cd in rice grains is mainly regulated by rice leaves, and the content of Cd accumulated in leaves directly affects Cd concentration in brown rice. In this study, the Cd contents in roots, culm, leaves and brown rice at the maturity stage were determined. Results showed that 100 mg/L ALA treatment could significantly reduce the Cd content in rice roots, culm and brown rice, indicating that this concentration of ALA could effectively inhibit Cd absorption and accumulation in the progress of Cd transport from shoots to grains, while a higher concentration of ALA had an opposite effect. In addition, 0.15 mg/L EBR treatment directly resulted in the significant increase of Cd accumulation in rice roots, culm, leaves and brown rice, while 0.30 mg/L exogenous EBR treatment reduced the Cd content in brown rice properly, but it was not significant. Although 0.30 mg/L EBR treatment obviously increased Cd concentration in culm, there were no significant differences in roots and leaves. The mechanism of EBR influenced the Cd concentration in brown rice might be affected by the progress of Cd long-distance transport from root to shoot. The physical and chemical properties of soil can directly affect the Cd absorption in rice. Usually, the total concentration of Cd in the soil is used to judge whether the soil is Cd-contaminated. However, what actually worked in the soil is the available Cd which directly affects the Cd absorption in rice (Römkens et al, 2009; Kim et al, 2016). At the same time, the rising of

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pH in the soil can decrease the bioavailability of heavy metals including Cd in rice (Vig et al, 2003). Bian et al (2016) found that in Cd-contaminated acidic soils, increasing the pH of the soil by adding Ca(OH)2 can directly change the available Cd content in the soil. Therefore, in this study, we measured soil pH, available Cd and total Cd to determine whether soil physicochemical properties would change under different treatment conditions. ALA and EBR treatments had no significant effects on soil pH and total Cd content, but significantly affected the available Cd content of the soils. Under 100 mg/L exogenous ALA treatment, the effective Cd content was significantly decreased compared with CK, while 200 mg/L ALA treatment increased the content of effective Cd in the soil. Therefore, ALA treatment could effectively change the speciation of Cd in soil. In addition, exogenous EBR treatment also had a certain effect on the speciation of soil Cd, which would increase the content of available Cd in the soil. ALA has been used as a plant growth regulator to alleviate damage caused by abiotic stress. Liu D et al (2011) found that ALA can both increase the contents of chlorophyll and carotenoids. Application with ALA enhances the net photosynthetic rate, accumulation of chlorophyll, and concentrations of intermediates related to chlorophyll (Xiong et al, 2018). Naeem et al (2010) indicated that treatment with ALA improves plant growth, photosynthetic gas exchange capacity and chlorophyll content under salinity stress. Photosynthesis plays an important role in the growth of plants, and a series of changes such as decreased rice biomass and slowed growth caused by Cd stress may be related to the inhibition of rice photosynthesis. This study showed that foliar application with ALA effectively reduced the toxic effect of Cd on rice, which may be due to that ALA promoted the photosynthesis, increased the metabolism and reduced the Cd accumulation in rice. Meanwhile, treatment with ALA improves the ultrastructures of chloroplast, mitochondria and nucleus, as well as increases the protein production related to stress response, which suggest that ALA is a potential plant growth regulator through alleviation of the physiological and ultrastructural changes (Xu et al, 2018). After treatment with different concentrations of ALA under Cd stress, foliar application of ALA significantly improves the Cd effect and the structure of mesophyll cells (Ali et al, 2013b). Naeem et al (2012) surveyed that ALA has a positive effect on ultrastructural

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changes under salinity stress. The ultrastructure of cell in rice plays an important role in Cd fixation. Foliar application with ALA affects the structure of chloroplasts and other cells that may lead to change of the Cd concentration which is fixed in each cell, and then directly leads to a decrease of Cd accumulation in different parts of rice plants. The addition of ALA significantly alleviates the environmental stress in plants by scavenging the reactive oxygen species (ROS) (Farid et al, 2018). Farid et al (2018) found that treatment with ALA reduces Chromium-induced toxicity in sunflower by enhancing plant antioxidant defense. Many antioxidant enzymes [ascorbate peroxidase (APX), peroxidase (POD), catalase (CAT) and superoxidase dismutase (SOD)] increases by the application of ALA on Cd-treated seedlings (Ali et al, 2013a). Liu D et al (2011) and Naeem et al (2011) revealed that activities of antioxidant enzymes are improved after treatment with ALA under drought and salinity stresses, respectively. Beyzaei et al (2015) showed a little higher SOD activity in the leaves of spinach after treatment with ALA. Similarly, they also found that ALA promotes the activity of glutathione reductase in spinach leaves. Under Cd stress, the oxidation balance would be broken, which would result in oxidative stress in rice. Our results showed that 100 mg/L ALA treatment could effectively reduce Cd accumulation in rice. Combined with previous results, we speculated that a major mechanism of ALA action on the agronomic traits of rice might be related to the coordinated response of oxidation equilibrium and photosynthesis in rice. Combined with various studies, the possible mechanisms of ALA to reduce Cd accumulation in rice were as follows: (1) Foliar application with ALA increased the production of chlorophyll and promoted the photosynthesis, then increased the metabolism of Cd during the growth of rice; (2) ALA changed the ultrastructure of rice, which had a certain influence on the transport progress and the fixed Cd content in certain parts, then alleviated the Cd accumulation in brown rice; (3) Foliar application with ALA could keep the oxidation balance, then reduced the toxic effects of Cd in rice. In the future, we will further elucidate its specific mechanism of rice response to Cd stress. In addition, BRs also could induce related-stress tolerance in plants such as low temperature and heavy metals (Bajguz and Hayat, 2009; Hayat S et al, 2010; Russinova, 2011). Similarly, the mechanism of EBR,

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which belongs to BRs, is also related to photosynthesis in plants in abiotic stress, and EBR plays a crucial role in protecting photosynthesis organizations from damage caused by high temperature stress (Zhang et al, 2013). Li et al (2012) studied the effect of EBR on chlorophyll (Chl) content and photosystem II (PS II) photochemistry in drought-stressed plants, and finally found Chl content and variable to maximum Chl fluorescence ratio (Fv/Fm) are higher in plants without EBR treatment. Cd stress could cause oxidative damage in rice. Many studies have shown that EBR plays an important role in alleviating the oxidative stress caused by the environment stress. EBR treatment alleviates oxidative damage through the up-regulation of antioxidative capacity under Zn stress (Ramakrishna and Rao, 2012; Yusuf et al, 2012). Foliar application with EBR could change the activity of antioxidative enzymes (SOD, POD, CAT and GR) and antioxidant molecules (glutathione and ascorbate) (Ding et al, 2012; Talaat and Shawky, 2013). Foliar application with EBR can also reduce the content of H2O2 in plants which is an important part of ROS (Ding et al, 2012). The specific protection mechanisms of EBR are speculated to the alleviated oxidative damage, the improved functioning of PS II (Janeczko et al, 2011), and the increased chlorophyll content, especially chlorophyll b (Yuan et al, 2012). Our results showed that after 0.15 mg/L EBR treatment, the accumulation of Cd in rice was increased, but treatment with 0.30 mg/L EBR alleviated the increasing trend of Cd accumulation in rice appropriately. Different concentrations of EBR had different effects on the physiological characteristics of rice, then induced the molecular mechanism of rice against Cd, and finally influenced the Cd accumulation in various parts of rice.

CONCLUTIONS Exogenous ALA treatment (100 mg/L) could effectively reduce the Cd accumulation of brown rice in moderate Cd contaminated paddy fields, which would be an effective technical method for the safe production of rice in Cd polluted farmland in the further. ALA treatment could adjust the available Cd of soil, which might due to the change of root exudates, physiological characteristics and molecular mechanism of rice induced by ALA, and the main process by which ALA affected Cd accumulation in brown rice was speculated to be the progress of Cd

WANG Feijuan, et al. Effects of Exogenous ALA and EBR Treatments on Cd Stressed Rice

transport from shoot to grain. Therefore, further research is needed on the mechanism of reduced Cd accumulation in brown rice under ALA treatment. Exogenous EBR treatment (0.15 mg/L) obviously increased the Cd content in roots, culm, leaves and brown rice, while 0.30 mg/L exogenous EBR treatment reduced the Cd content in brown rice properly. Furthermore, 0.30 mg/L EBR obviously increased Cd content in culm while there were no significant differences in roots and leaves. The mechanism of EBR influenced the Cd content of brown rice might be the progress of Cd absorption and long-distance transport from root to shoot. Hence, in the follow-up study, we will further explore the appropriate EBR concentration to study whether it can reduce the Cd content in brown rice, and elucidate the specific molecular physiological mechanism that affects the Cd accumulation in rice.

ACKNOWLEDGEMENTS This work was supported by the Natural Science Foundation of Zhejiang Province, China (Grant No. LY17C020005), the Key Research and Development Project of Zhejiang Province, China (Grant No. 2015C03020-4), the National Nature Science Foundation of China (Grant No. 31401356), Jinhua Science and Technology Project (Grant No. 2015-2012), and the National Training Program for College Students to Innovate and Start Enterprise (Grant No. 201710356013).

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